Novel polypeptide

ABSTRACT

The present invention provides a novel polypeptide having a β1,3-N-acetylglucosaminyltransferase activity, an agent for synthesizing a sugar chain comprising the polypeptide, a process for producing a sugar chain or a complex carbohydrate using the agent for synthesizing a sugar chain, DNA encoding the polypeptide, a process for producing the polypeptide, an antibody against the polypeptide, and a diagnosis method and a medicament for treatment for inflammation, cancer or tumor metastasis using the DNA or the antibody. The present invention is useful for synthesis of a useful sugar chain and diagnosis and treatment for inflammatory diseases, cancer or tumor metastasis.

TECHNICAL FIELD

[0001] The present invention relates to a novel polypeptide having alactosylceramide β1,3-N-acetylglucosaminyltransferase activity and aparagloboside β1,3-N-acetylglucosaminyltransferase activity; an agentfor synthesizing a sugar chain, which comprises the polypeptide as anactive ingredient; a DNA encoding the polypeptide, an agent fordetecting inflammation, cancer or tumor metastasis, which comprises theDNA; a recombinant DNA obtainable by inserting the DNA into a vector; atransformant comprising the recombinant DNA; a process for producing thepolypeptide using the transformant; a process for producing a sugarchain or complex carbohydrate using the polypeptide; a process forproducing a sugar chain or complex carbohydrate using the transformant;a method for detecting inflammation, cancer or tumor metastasis using anoligonucleotide obtainable from a DNA encoding the polypeptide; anantibody which recognizes the polypeptide; a method forimmunohistostaining using the antibody; an agent for immunohistostainingor an agent for diagnosing inflammatory disease, cancer or tumormetastasis, which comprises the antibody; a medicament comprising thepolypeptide, the DNA, the recombinant vector or the antibody; a methodfor screening a compound which changes a lactosylceramideβ1,3-N-acetylglucosaminyltransferase activity and a paraglobosideβ1,3-N-acetylglucosaminyltransferase activity of the polypeptide; amethod for screening a compound which changes expression of the gene; apromoter DNA which controls transcription of the gene; a method forscreening a compound which changes efficiency of transcription by thepromoter DNA; a compound obtainable by the screening methods; anon-human knockout animal the gene is deleted or mutated; and the like.

BACKGROUND ART

[0002] Lactosylceramide β1,3-N-acetylglucosaminyltransferase is anenzyme having an activity to transfer N-acetylglucosamine viaβ1,3-linkage to a galactose residue present in the non-reducing terminalof lactosylceramide (Galβ1-4Glc-ceramide). Neolacto-series glycolipids,lacto-series glycolipids, ganglio-series glycolipids, globo-seriesglycolipids and isoglobo-series glycolipids are synthesized from thelactosylceramide (Galβ1-4Glc-ceramide), and lactosylceramideβ1,3-N-acetylglucosaminyltransferase is a key enzyme of the synthesis ofneolacto-series glycolipids and lacto-series glycolipids.

[0003] Ganglioside GM3 (NeuAcα2-3Galβ1-4Glc-ceramide) is synthesizedwhen GM3 synthase acts upon lactosylceramide. AsialoGM2(GalNAcβ1-4Galβ1-4Glc-ceramide) is synthesized when GM2 synthase actsupon lactosylceramide. Since many other gangliosides are synthesizedfrom GM3 and asialoGM2, GM3 synthase and GM2 synthase can be regarded askey enzymes of the synthesis of ganglio-series glycolipids. On the otherhand, when lactosylceramide α1,4-galactosyltransferase acts uponlactosylceramide, Galα1-4Galβ1-4Glc-ceramide is synthesized and then aseries of globo-series glycolipids are synthesized. Whenlactosylceramide α1,3-galactosyltransferase acts upon lactosylceramide,Galα1-3Galβ1-4Glc-ceramide is synthesized and then a series ofisoglobo-series glycolipids are synthesized.

[0004] Accordingly, it can be said that lactosylceramideα1,4-galactosyltransferase and lactosylceramideα1,3-galactosyltransferase are key enzymes of the synthesis ofglobo-series glycolipids and isoglobo-series glycolipids, respectively.It is considered that synthesis of a specific glycolipid in a cell iscontrolled by the expression and expression level of the above keyenzymes.

[0005] Neolacto-series glycolipid is a glycolipid havingGalβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide backbone, and lacto-series.glycolipid is a glycolipid having a Galβ1-3GlcNAcβ1-3Galβ1-4Glc-ceramidebackbone. Examples of the neolacto-series glycolipid includeparagloboside (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide),sialylparagloboside (NeuAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide),NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-ceramide and the like.Examples of the lacto-series glycolipid includeGalβ1-3GlcNAcβ1-3Galβ1-4Glc-ceramide,NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-ceramide,NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc-ceramide and the like.

[0006] It has been found that lacto- or neolacto-series glycolipids towhich fucose and sialic acid are added are accumulated in large amountsin many human cancers (particularly colon cancer or gastric cancer)[Annu. Rev. Immunol., 2, 103 (1984), Chem. Phys. Lipids, 42, 209(1986)]. As a result of the measurement of glycosyltransferase activityin colon cancer tissues and their peripheral normal tissues or variouscolon cancer cell lines, it has been found that the activity oflactosylceramide β1,3-N-acetylglucosaminyltransferase is increased incolon cancer tissues and various colon cancer cell lines [J. Biol.Chem., 262, 15649 (1987)]. This result suggests that increase of thelacto- or neolacto-series glycolipids in colon cancer is caused by theincreased lactosylceramide β1,3-N-acetylglucosaminyltransferaseactivity.

[0007] When a human promyelocytic cell line, HL-60, is treated withdimethyl sulfoxide or retinoic acid, it differentiates into granulocytecells. On the other hand, when HL-60 is treated with phorbol ester suchas phorbol-12-myristate-13-acetate (PMA), it differentiates intomonocyte/macrophage. While neolacto-series glycolipids (paraglobosideand sialylparagloboside) increase and ganglioside GM3 decreases when itis differentiated into granulocyte cells, ganglioside GM3 increases andneolacto-series glycolipids decrease when it is differentiated intomonocyte/macrophage. Also, when HL-60 is cultured by adding aneolacto-series glycolipid, it differentiates into granulocyte cells,and when HL-60 is cultured by adding ganglioside GM3, it differentiatesinto monocyte/macrophage. The results show that expression of a specificglycolipid is important in determining the induction and direction ofthe differentiation. When HL-60 is treated with retinoic acid, the GM3synthase activity does not change but the lactosylceramideβ1,3-N-acetylglucosaminyltransferase activity increases [J. Biol. Chem.,267, 23507 (1992)]. Thus, it is considered that, in the HL-60 treatedwith retinoic acid, increase of neolacto-series glycolipids and decreaseof ganglioside GM3 are induced caused by the increased lactosylceramideβ1,3-N-acetylglucosaminyltransferase activity, and it differentiatesinto granulocyte cells as the result. On the other hand, when HL-60 istreated with PMA, the GM3 synthase activity increases and thelactosylceramide β1,3-N-acetylglucosaminyltransferase activity decreases[J. Biol. Chem., 267, 23507 (1992)].

[0008] Accordingly, it is considered that, in the HL-60 treated withPMA, increase of ganglioside GM3 and decrease of neolacto-seriesglycolipids are caused by the increased GM3 synthase activity and thereduced lactosylceramide β1,3-N-acetylglucosaminyltransferase activity,and it differentiates into monocyte/macrophage as the result. It isconsidered that lactosylceramide β1,3-N-acetylglucosaminyltransferaseand GM3 synthase are taking an important role in determining theinduction and direction of the differentiation of promyelocyte.

[0009] It is known that leukocytes express different glycolipidsdepending on their types and differentiation stages. For example, maturemyelogenous cell expresses only neutral neolacto-series glycolipid [Mol.Cell. Biochem., 47, 81 (1982), J. Biol. Chem., 260, 1067 (1985)]. On theother hand, mature lymphocyte expresses only globo-series glycolipid[Mol. Cell. Biochem., 47, 81 (1982)]. It is suggested based on ananalysis using leukocyte cell lines that the above differences ofglycolipids are due to difference in the lactosylceramideβ1,3-N-acetylglucosaminyltransferase activity. It has been found thatthe lactosylceramide β1,3-N-acetylglucosaminyltransferase activity isdetected in myelogenous cell lines such as K-562, KG-1 and HL-60, butthis enzyme activity is not detected in lymphocyte cell lines such asReh, CCRF-CEM, MOLT-4, Ramos and RPMI 8226 [Archives of Biochemistry andBiophysics, 303, 125 (1993)].

[0010] It is known that a glycolipid having 3-sulfoglucuronic acid onthe non-reducing terminal of its sugar chain (e.g.,SO₄3GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide) is expressed at aspecific period of time and in a specific region during thedifferentiation of nerve system. It has been suggested that thisglycolipid is concerned in the mutual recognition of nerve cells andmigration of nerves [J. Biol. Chem., 273, 8508 (1998)]. Since expressionof the 3-sulfoglucuronic acid-containing glycolipid(SO₄3GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide) in nerve cells iscontrolled by lactosylceramide β1,3-N-acetylglucosaminyltransferase, itis considered that mutual recognition and migration of nerve cells arecontrolled by the expression of lactosylceramideβ1,3-N-acetylglucosaminyltransferase [J. Biol. Chem., 273, 8508 (1998)].Since 3-sulfoglucuronic acid is recognized also by monoclonal antibodyHNK-1 for a marker of human NK cell, it is also called HNK-1 epitope.Thus, it is considered that the 3-sulfoglucuronic acid-containingglycolipid plays an important role in the function of NK cell.

[0011] A sugar chain having GlcNAcβ1-3Gal structure is present in sugarchains of neolacto- and lacto-series glycolipids and also in N-linkedsugar chains and O-linked sugar chains of glycoproteins, and inoligonsaccharide. For example, lacto-N-neotetraose(Galβ1-4GlcNAcβ1-3Galβ1-4Glc) and lacto-N-tetraose(Galβ1-3GlcNAcβ1-3Galβ1-4Glc), which exist in human milk, or variousoligosaccharides having them as backbones can be cited as theoligosaccharides having GlcNAcβ1-3Gal structure [Acta Paediatrica, 82,903 (1993)]. The GlcNAcβ1-3Gal structure is also an element constitutinga poly-N-acetyllactosamine sugar chain. The poly-N-acetyllactosaminesugar chain is a sugar chain having structure in whichN-acetyllactosamine is repeatedly bound via β1,3-linkage[(Galβ1-4GlcNAcβ1-3)_(n); n is 2 or more], which is present in N-linkedsugar chains and O-linked sugar chains of glycoproteins and also presentin glycolipid sugar chains and oligosaccharides. Whether or notlactosylceramide β1,3-N-acetylglucosaminyltransferase uses substratesother than lactosylceramide, such as paragloboside, N-linked sugarchains and O-linked sugar chains of glycoproteins or oligosaccharides,has not been found.

[0012] Up to date, the lactosylceramideβ1,3-N-acetylglucosaminyltransferase activity has been detected in colontissues, colon cancer tissues, colon cancer cell lines (Colo205, SW403and the like) and myeloid cell lines (K-562, KG-1 and HL-60), but thereare no reports on the high purity purification of lactosylceramideβ1,3-N-acetylglucosaminyltransferase [J. Biol. Chem., 262, 15649 (1987),Archives of Biochemistry and Biophysics, 260, 461 (1988), CarbohydrateResearch, 209, 261 (1991), Archives of Biochemistry and Biophysics, 303,125 (1993)].

[0013] On the other hand, regarding enzymes having the activity totransfer N-acetylglucosamine via β1,3-linkage to the galactose residuepresent in the non-reducing terminal of sugar chains (hereinafterreferred to as “Gal 1,3-N-acetylglucosaminyltransferase”), there arereports on their partial purification but it is not clear whether theseenzymes use lactosylceramide as a substrate [J. Biol. Chem., 268, 27118(1993), J. Biol. Chem., 267, 2994 (1992), J. Biol. Chem., 263, 12461(1988), Jpn. J. Med. Sci. Biol., 42, 77 (1989)].

[0014] Regarding cloning of genes, genes of two types of Galβ1,3-N-acetylglucosaminyltransferases have so far been cloned [Proc.Natl. Acad. Sci. USA, 94, 14294-14299 (1997), Proc. Natl. Acad. Sci.USA, 96, 406-411 (1999)]. It has been shown that β3GnT as one of themuses paragloboside as its substrate in vitro, but its activity is weakwhen lactosylceramide is used as the substrate [Glycobiology, 9, 1123(1999)]. Also, it has not been found whether β3GnT uses lactosylceramideand paragloboside as its substrates inside cells. In addition, thepresence of the other Gal β1,3-N-acetylglucosaminyltransferase is notclear.

[0015] Since a large number of sugar chains having the GlcNAcβ1-3Galstructure are present, it seems highly possible that two or more Galβ1,3-N-acetylglucosaminyltransferases having different acceptorspecificity and expression tissue are present and have respectivedifferent functions. Accordingly, it is considered that lactosylceramideβ1,3-N-acetylglucosaminyltransferase can be identified by cloning a Galβ1,3-N-acetylglucosaminyltransferase which is different from the two Galβ1,3-N-acetylglucosaminyltransferases so far cloned, and examining itsacceptor specificity.

[0016] As described above, it is known that lacto-N-neotetraose(Galβ1-4GlcNAcβ1-3Galβ1-4Glc) and lacto-N-tetraose(Galβ1-3GlcNAcβ1-3Galβ1-4Glc) or various oligosaccharides having them asbackbones are present in human milk [Acta Paediatrica, 82, 903 (1993)].These oligosaccharides have the GlcNAcβ1-3Gal structure in common. It isconsidered that they have a function to prevent babies from infectionwith viruses and microorganisms and a function to neutralize toxins.Also, they have an activity to accelerate growth of Lactobacillusbifidus which is a beneficial enteric bacterium. On the other hand,kinds of oligosaccharide existing in the milk of animals such as cowsand mice are few and mostly lactose, and the above oligosaccharidesexisting in human milk are hardly present therein.

[0017] It may be industrially markedly useful if the aboveoligosaccharides contained in human milk or a milk containing them canbe produced efficiently. When the gene of a Galβ1,3-N-acetylglucosaminyltransferase involved in the synthesis of theabove oligosaccharides contained in human milk can be obtained, it ispossible to use it in the efficient synthesis of the aboveoligosaccharides, but the enzyme has not been found yet.

[0018] Among sugar chains having the GlcNAcβ1-3Gal structure,particularly poly-N-acetyllactosamine sugar chain is a backbone sugarchain of many functional sugar chains (selectin ligand sugar chains,receptor sugar chains for microorganisms and viruses, SSEA-1 sugarchains, cancer-related sugar chains and the like) and deeply related toembryogenesis, cell differentiation or diseases such as inflammation andcancer. The poly-N-acetyllactosamine sugar chain also plays an importantrole in the stabilization of glycoprotein.

[0019] Since there is a possibility that Galβ1,3-N-acetylglucosaminyltransferases involved in the synthesis ofpoly-N-acetyllactosamine sugar chain functioning in respective cases aredifferent, there is a possibility that a Galβ1,3-N-acetylglucosaminyltransferase different from the two enzymes sofar cloned exists. There is a possibility that lactosylceramideβ1,3-N-acetylglucosaminyltransferase is related to the synthesis ofpoly-N-acetyllactosamine sugar chain by transferring N-acetyllactosamineto sugar chains having Galβ1-4Glc or Galβ1-4GlcNAc at the non-reducingterminal (e.g., paragloboside) in addition to lactosylceramide.

[0020] Synthesis, function and application of thepoly-N-acetyllactosamine sugar chain are described below.

[0021] The poly-N-acetyllactosamine sugar chain is synthesized by themutual actions of a GlcNAc β1,4-galactosyltransferase (an enzyme havingan activity to transfer galactose via β1,4-linkage to theN-acetylglucosamine residue present in the non-reducing terminal ofsugar chains) and Gal β1,3-N-acetylglucosaminyltransferase. RegardingGlcNAc β1,4-galactosyltransferase, genes of four enzymes (β4Gal-T1,β4Gal-T2, β4Gal-T3 and β4Gal-T4) have so far been cloned, and acceptorspecificity of each enzyme has been analyzed [J. Biol. Chem., 2,31979-31991 (1997), J. Biol. Chem., 273, 29331-29340 (1997)].

[0022] Saccharides such as fucose, sialic acid, N-acetylgalactosamineand galactose, a sulfate group and the like are added to linear orbranched poly-N-acetyllactosamine sugar chains to thereby form variouscell-specific or stage-specific sugar chains (functional sugar chains,blood group sugar chains, cancer-related sugar chains and the like)[Glycobiology Series, (1) to (6), edited by Akira Kobata, SenitirohHakomori and Yoshitaka Nagai, published by Kodansha (1993)].

[0023] It is known that poly-N-acetyllactosamine sugar chains having asialyl Lewis x sugar chain [NeuAcα2-3Galβ1-4(fucal-3)GlcNAc] at theirterminal are present on granulocytes, monocytes or activated T cells,and it is considered that these sugar chains relate to the accumulationof the leukocytes into inflammatory regions by functioning as ligands ofadhesion molecules, E-selectin and P-selectin [Glycobiology Series, (1)to (6), edited by Akira Kobata, Senitiroh Hakomori and Yoshitaka Nagai,published by Kodansha (1993)].

[0024] It is also known that poly-N-acetyllactosamine sugar chainshaving a sialyl Lewis x sugar chain and a sialyl Lewis a sugar chain[NeuAcα2-3Galβ1-3(fucα1-4)GlcNAc] at the terminal are present on cancercells such as colon cancer, and it is suggested that the sugar chainsare also involved in tumor metastasis by functioning as ligands ofE-selectin and P-selectin [Glycobiology Series, (1) to (6), edited byAkira Kobata, Senitiroh Hakomori and Yoshitaka Nagai, published byKodansha (1993)].

[0025] It is known that the structure of the poly-N-acetyllactosaminesugar chain changes during the process of embryogenesis, celldifferentiation or malignant transformation of cells [GlycobiologySeries, (1) to (6), edited by Akira Kobata, Senitiroh Hakomori andYoshitaka Nagai, published by Kodansha (1993)). While a linearpoly-N-acetyllactosamine sugar chain is expressed on human fetalerythrocytes, a branched poly-N-acetyllactosamine sugar chain isexpressed on adult erythrocytes [Glycobiology Series, (1) “Various Worldof Sugar Chains”, edited by Akira Kobata, Senitiroh Hakomori andYoshitaka Nagai, published by Kodansha, 1993]. ABO blood type antigensare expressed at the termini of the poly-N-acetyllactosamine sugarchains on erythrocytes. When a blood type antigen is expressed atrespective termini of branched poly-N-acetyllactosamine sugar chains, itbecomes a multivalent antigen and its binding ability with antibodiesfor blood type sugar chains increases 10³ times or more in comparisonwith a linear type antigen.

[0026] It is known that a series of sugar chain antigens aresystemically expressed during the developing stage of mouse earlyembryo. SSEA-1 (stage specific embryonic antigen-1) is a Lewis x sugarchain [Galβ1-4(fucα1-3)GlcNAc] existing at the terminal of apoly-N-acetyllactosamine sugar chain, and expression of the antigenstarts at the 8-cell stage, reaches its peak at the morula stage andgradually disappears after the blastocyst stage [Glycobiology Series,(3) “Glycobiology of Cell Society”, edited by Akira Kobata, SenitirohHakomori and Yoshitaka Nagai, published by Kodansha, 1993]. The morulastage corresponds to a shifting stage in which germ cells so farproliferated by repeating simple numerical increase by cell divisionshift for the first time to the stage of blastocyst havingdifferentiated “shape”. Just before forming blastocyst, the morula cellsclosely assemble and cause cell compaction. When an oligosaccharidehaving the SSEA-1 is added, this cell compaction is inhibited and normaldevelopment thereafter is also inhibited [J. Exp. Med., 160, 1591(1984)]. It is also known that adhesion of mouse teratocarcinoma isinhibited by an anti-SSEA-1 antibody [Glycobiology Series, (3)“Glycobiology of Cell Society”, edited by Akira Kobata, SenitirohHakomori and Yoshitaka Nagai, published by Kodansha, 1993]. The abovefindings show that the SSEA-1 plays an important role in the developmentof early embryo by acting as an adhesion molecule or a sugar chainsignal.

[0027] It is known that poly-N-acetyllactosamine sugar chains areexpressed in a large quantity in cancer cells in comparison withcorresponding normal cells [J. Biol. Chem., 259, 10834 (1984), J. Biol.Chem., 261, 10772 (1986), J. Biol. Chem., 266, 1772 (1991), J. Biol.Chem., 267, 5700 (1992)]. It is known that when N-ras proto-oncogene isexpressed in NIH3T3 cell, molecular weight of N-linked sugar chain onthe cell surface is increased and the cell acquires infiltratingability, and at the same time, the amount of poly-N-acetyllactosaminesugar chain in the N-linked sugar chain is increased and activities ofβ1,4-galactosyltransferase and β1,3-N-acetylglucosaminyltransferasewhich relates to the synthesis of poly-N-acetyllactosamine sugar chainare also increased [J. Biol. Chem., 266, 21674 (1991)].

[0028] Galectins are a group of lectins having affinity forβ-galactoside, which relate to the adhesion and signal transduction ofcells, and their relation to diseases such as cancer is also suggested[Trends in Glycoscience and Glycotechnology, 9, 9 (1997)]. To date, 10types of galectins are known in mammals. It is known that among these,galectin-1 and galectin-3 bind to linear poly-N-acetyllactosamine sugarchains with high affinity, and it is considered that certainglycoproteins containing these sugar chains are ligands of thesegalectins [Trends in Glycoscience and Glycotechnology, 9, 9 (1997),Trends in Glycoscience and Glycotechnology, 9, 47 (1997)].

[0029] Poly-N-acetyllactosamine sugar chains having sialic acids addedto their termini serve as receptors for Mycoplasma and microorganisms[Acta Paediatrica, 82, 903 (1993)].

[0030] Thus, poly-N-acetyllactosamine sugar chains form backbone sugarchains of many functional sugar chains (selectin ligand sugar chains,receptor sugar chains for microorganisms and viruses, SSEA-1 sugarchain, cancer-related sugar chains and the like) and blood type sugarchains, and play important roles in efficiently presenting the sugarchains.

[0031] It is expected that poly-N-acetyllactosamine sugar chains havingsialyl Lewis x sugar chains will become a medicament havinganti-inflammatory effect or tumor metastasis inhibitory effect, as aselectin antagonist.

[0032] It is known that an oligosaccharide in which multivalent (four)sialyl Lewis x sugar chains (tetrasaccharides) is linked topoly-N-acetyllactosamine sugar chains shows the activity as a selectinantagonist at a low concentration of {fraction (1/100)} or less incomparison with non-multivalent sialyl Lewis x sugar chains(tetrasaccharides) [J. Exp. Med., 182, 1133 (1995), Glycobiology, 6, 65(1996), Glycobiology, 7, 453 (1997), Eur. J. Immunol., 27, 1360 (1997)].Although a partially purified β1,3-N-acetylglucosaminyltransferase hasbeen used for the synthesis of the poly-N-acetyllactosamine sugar chainmoiety of the oligosaccharides, supply of this enzyme is a limitingfactor so that it is difficult to synthesize a large amount ofpoly-N-acetyllactosamine sugar chains [Glycobiology, 7, 453 (1997)].

[0033] On the other hand, it is possible to synthesizepoly-N-acetyllactosamine sugar chains by chemical synthesis, but itssynthesis requires markedly complex steps [Tetrahedron Letter, 24, 5223(1997)].

[0034] Accordingly, an efficient method for synthesizingpoly-N-acetyllactosamine sugar chains is expected. Although the twotypes of Gal β1,3-N-acetylglucosaminyltransferases and their genes sofar cloned may be used, it is considered that the use of other Galβ1,3-N-acetylglucosaminyltransferase having different substratespecificity and functions (e.g., lactosylceramideβ1,3-N-acetylglucosaminyltransferase) and its gene may be efficient insome cases depending on the purpose.

[0035] The poly-N-acetyllactosamine sugar chains are also important forthe stabilization of glycoprotein. Lysosome associated membraneglycoprotein-1 (lamp-1) and lysosome associated membrane glycoprotein-2(lamp-2) are glycoproteins which exist in lysosome (partly exist on thecell surface) and almost completely cover inner face of lysosomemembrane. Many sugar chains (some of them containing apoly-N-acetyllactosamine sugar chain) are added to lamp-1 and lamp-2 toprevent degradation of lamp-1 and lamp-2 by hydrolases in lysosome. Itis known that when a human promyelocyte cell line HL-60 is treated withdimethyl sulfoxide, it differentiates into granulocyte cells, and duringthis differentiation process, the number of poly-N-acetyllactosaminesugar chains added to lamp-1 and lamp-2 increases and the metabolic rate(degradation rate) of lamp-1 and lamp-2 decreases [J. Biol. Chem., 265,20476 (1990)].

[0036] Examples for increase of the ability to synthesizepoly-N-acetyllactosamine sugar chains are shown below.

[0037] It is shown that poly-N-acetyllactosamine sugar chains are addedto sugar chains of cell membrane glycoproteins when F9 cell is treatedwith retinoic acid or when Swiss 3T3 cell is treated with TGF-β [J.Biol. Chem., 268, 1242 (1993), Biochim. Biophys. Acta., 1221, 330(1994)].

[0038] It is known that activities of β1,4-galactosyltransferase andβ1,3-N-acetylglucosaminyltransferase involved in the synthesis ofpoly-N-acetyllactosamine sugar chains are increased, and the amount ofpoly-N-acetyllactosamine sugar chains in N-binding type sugar chains ofglycoprotein is increased, when N-ras proto-oncogene is expressed inNIH3 T3 cells [J. Biol. Chem., 266, 21674 (1991)]. The molecular weightof a cell surface membrane protein CD43, CD45 or CD44 is increased whena core 2 β1,6-N-acetylglucosaminyltransferase gene is expressed in aT-cell line EL-4 [J. Biol. Chem., 271, 18732 (1996)]. The reason forthis may be that sugar chains synthesized by the core 2β1,6-N-acetylglucosaminyltransferase become a good substrate ofβ1,3-N-acetylglucosaminyltransferase involved in the synthesis ofpoly-N-acetyllactosamine sugar chains.

[0039] Also, it is known that the amount of poly-N-acetyllactosaminesugar chains added to lamp-1 or lamp-2 is increased when HL-60 cells arecultured at 27° C. [J. Biol. Chem., 266, 23185 (1991)].

[0040] However, there are no reports to date on the efficient productionof recombinant glycoproteins to which poly-N-acetyllactosamine sugarchains are added, in host cells suitable for the production ofrecombinant glycoproteins (e.g., Namalwa cell, Namalwa KJM-1 cell, CHOcell). Accordingly, development of a process for efficiently producing arecombinant glycoprotein to which poly-N-acetyllactosamine sugar chainsare added is an industrially important subject.

[0041] Although the two types of Galβ1,3-N-acetylglucosaminyltransferases so far cloned can be used, it isconsidered that use of other Gal β1,3-N-acetylglucosaminyltransferasehaving different substrate specificity and functions (e.g.,lactosylceramide β1,3-N-acetylglucosaminyltransferase) may be efficientin some cases depending on the purpose.

DISCLOSURE OF THE INVENTION

[0042] An object of the present invention is to provide synthesis ofuseful sugar chains; medicaments such as antiinflammatory agents,anti-infective agents and tumor metastasis inhibitory agents; foods suchas dairy products; a method for improving protein stability, and thelike; and a method for diagnosing inflammatory diseases and types andmalignancies of cancers, by use of a novel polypeptide having aβ1,3-N-acetylglucosaminyltransferase activity.

[0043] The present invention relates to the following (1) to (79).

[0044] (1) A polypeptide which comprises the amino acid sequencerepresented by SEQ ID NO:1.

[0045] (2) A polypeptide which comprises an amino acid sequence ofpositions 39 to 378 in the amino acid sequence represented by SEQ IDNO:1.

[0046] (3) A polypeptide which comprises an amino acid sequence in whichat least one amino acid in the amino acid sequence in the polypeptideaccording to (1) or (2) is deleted, substituted or added, and has aβ1,3-N-acetylglucosaminyltransferase activity.

[0047] (4) A polypeptide which comprises an amino acid sequence having60% or more homology with the amino acid sequence in the polypeptideaccording to (1) or (2), and has a β1,3-N-acetylglucosaminyltransferaseactivity.

[0048] (5) The polypeptide according to (3) or (4), wherein theβ1,3-N-acetylglucosaminyltransferase activity is an activity to transferN-acetylglucosamine via β1,3-linkage to a galactose residue present inits non-reducing terminal of a sugar chain.

[0049] (6) The polypeptide according to any one of (3) to (5), whereinthe β1,3-N-acetylglucosaminyltransferase activity is an activity totransfer N-acetylglucosamine via β1,3-linkage to a galactose residuepresent in its non-reducing terminal of a sugar chain of an acceptorselected from i) galactose, N-acetyllactosamine (Galβ1-4GlcNAc),Galβ1-3GlcNAc or lactose (Galβ1-4Glc), ii) an oligosaccharide havinggalactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure inits non-reducing terminal, and iii) a complex carbohydrate having agalactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure inits non-reducing terminal.

[0050] (7) The polypeptide according to (6), wherein the complexcarbohydrate having galactose, N-acetyllactosamine, Galβ1-3GlcNAc orlactose structure in its non-reducing terminal is lactosylceramide(Galβ1-4Glc-ceramide) or paragloboside(Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide).

[0051] (8) The polypeptide according to (6) or (7), wherein the complexcarbohydrate is a complex carbohydrate selected from a glycoprotein, aglycolipid, a proteoglycan, a glycopeptide, a lipopolysaccharide, apeptidoglycan and a glycoside in which a sugar chain is linked to asteroid compound.

[0052] (9) A sugar chain synthesizing agent which comprises thepolypeptide according to any one of (1) to (8) as an active ingredient.

[0053] (10) A DNA which encodes the polypeptide according to any one of(1) to (8).

[0054] (11) A DNA which comprises the nucleotide sequence represented bySEQ ID NO:2.

[0055] (12) A DNA which comprises a nucleotide sequence of positions 135to 1268 in the nucleotide sequence represented by SEQ ID NO:2.

[0056] (13) A DNA which comprises a nucleotide sequence of positions 249to 1268 in the nucleotide sequence represented by SEQ ID NO:2.

[0057] (14) A DNA which hybridizes with a DNA comprising a nucleotidesequence complementary to the nucleotide sequence in the DNA accordingto any one of (10) to (13) under stringent conditions, and encodes apolypeptide having a β1,3-N-acetylglucosaminyltransferase activity.

[0058] (15) The DNA according to (14), wherein theβ1,3-N-acetylglucosaminyltransferase activity is an activity to transferN-acetylglucosamine via β1,3-linkage to a galactose residue present inits non-reducing terminal of a sugar chain.

[0059] (16) The DNA according to (14) or (15), wherein theβ1,3-N-acetylglucosaminyltransferase activity is an activity to transferN-acetylglucosamine via β1,3-linkage to a galactose residue present inits non-reducing terminal of a sugar chain of an acceptor selected fromi) galactose, N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc orlactose (Galβ1-4Glc), ii) an oligosaccharide having galactose,N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure in itsnon-reducing terminal, and iii) a complex carbohydrate having galactose,N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure in itsnon-reducing terminal.

[0060] (17) The DNA according to (16), wherein the complex carbohydratehaving galactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactosestructure in its non-reducing terminal is lactosylceramide orparagloboside.

[0061] (18) The DNA according to (16) or (17), wherein the complexcarbohydrate is a complex carbohydrate selected from a glycoprotein, aglycolipid, a proteoglycan, a glycopeptide, a lipopolysaccharide, apeptidoglycan and a glycoside in which a sugar chain is linked to asteroid compound.

[0062] (19) A DNA which comprises a nucleotide sequence complementary tothe nucleotide sequence in the DNA according to any one of (10) to (18).

[0063] (20) A recombinant vector which is obtainable by inserting theDNA according to any one of (10) to (18) into a vector.

[0064] (21) A recombinant vector which is obtainable by inserting an RNAcomprising a sequence homologous to the DNA according to any one of (10)to (18) into a vector.

[0065] (22) A transformant which comprises the recombinant vectoraccording to (20) or (21).

[0066] (23) The transformant according to (22), wherein the transformantis a transformant selected from the group consisting of a microorganism,an animal cell, a plant cell and an insect cell.

[0067] (24) The transformant according to (23), wherein themicroorganism is a microorganism belonging to the genus Escherichia.

[0068] (25) The transformant according to (23), wherein the animal cellis an animal cell selected from the group consisting of a mouse myelomacell, a rat myeloma cell, a mouse hybridoma cell, a CHO cell, a BHKcell, an African green monkey kidney cell, a Namalwa cell, a NamalwaKJM-1 cell, a human fetal kidney cell and a human leukemia cell.

[0069] (26) The transformant according to (23), wherein the plant cellis a plant cell selected from the group consisting of plant cells oftobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice plant,wheat, barley, rye, corn or flax.

[0070] (27) The transformant according to (23), wherein the insect cellis an insect cell selected from the group consisting of Spodopterafrugiperda ovarian cells, Trichoplusia ni ovarian cells and silkwormovarian cells.

[0071] (28) The transformant according to (22), wherein the transformantis a non-human transgenic animal or transgenic plant.

[0072] (29) A process for producing a polypeptide, which comprisesculturing the transformant according to any one of (22) to (27) in amedium to produce and accumulate the polypeptide according to any one of(1) to (8) in the culture, and recovering the polypeptide from theculture.

[0073] (30) A process for producing a polypeptide, which comprisesbreeding the non-human transgenic animal according to (28) to produceand accumulate the polypeptide according to any one of (1) to (8) in theanimal, and recovering the polypeptide from the animal.

[0074] (31) The process according to (30), wherein the accumulation iscarried out in animal milk.

[0075] (32) A process for producing a polypeptide, which comprisescultivating the transgenic plant according to (28) to produce andaccumulate the polypeptide according to any one of (1) to (8) in theplant, and recovering the polypeptide from the plant.

[0076] (33) A process for producing a polypeptide, which comprisessynthesizing the polypeptide according to any one of (1) to (8) by an invitro transcription-translation system using the DNA according to anyone of (10) to (18).

[0077] (34) A process for producing a sugar chain or complexcarbohydrate by using the sugar chain synthesizing agent according to(9) as an enzyme source, which comprises allowing

[0078] a) the enzyme source,

[0079] b) an acceptor selected from i) lactosylceramide(Galβ1-4Glc-ceramide) or paragloboside(Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide), ii) galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose(Galβ1-4Glc), iii) an oligosaccharide having galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose structurein its non-reducing terminal, and iv) a complex carbohydrate havinggalactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure inits non-reducing terminal, and

[0080] c) N-acetylglucosamine uridine 5′-diphosphate (UDP-GlcNAc) to bepresent in an aqueous medium to produce and accumulate a sugar chain orcomplex carbohydrate in which N-acetylglucosamine is added viaβ1,3-linkage to a galactose residue of the acceptor in the aqueousmedium, and recovering the sugar chain or complex carbohydrate from theaqueous medium.

[0081] (35) A process for producing a galactose-added sugar chain orcomplex carbohydrate by using the N-acetylglucosamine-added sugar chainor complex carbohydrate obtained by the process according to (34) as anacceptor, which comprises allowing

[0082] a) the acceptor,

[0083] b) GlcNAc β1,4-galactosyltransferase, and

[0084] c) uridine 5′-diphosphate galactose (UDP-Gal) to be present in anaqueous medium to produce and accumulate a reaction product in whichgalactose is added via β1,4-linkage to an N-acetylglucosamine residue atthe non-reducing terminal of the acceptor in the aqueous medium, andrecovering the galactose-added sugar chain or complex carbohydrate fromthe aqueous medium.

[0085] (36) A process for producing a poly-N-acetyllactosamine sugarchain-added sugar chain or complex carbohydrate by using the sugar chainsynthesizing agent according to (9) as an enzyme source, which comprisesallowing

[0086] a) the enzyme source,

[0087] b) GlcNAc β1,4-galactosyltransferase,

[0088] c) an acceptor selected from i) lactosylceramide(Galβ1-4Glc-ceramide) or paragloboside(Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide), ii) galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose(Galβ1-4Glc), iii) an oligosaccharide having galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose(Galβ1-4Glc) structure in its non-reducing terminal, iv) a complexcarbohydrate having galactose, N-acetyllactosamine, Galβ1-3GlcNAc orlactose structure in its non-reducing terminal and v) a sugar chain orcomplex carbohydrate obtained by the process according to (34) or (35),

[0089] d) uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc), and

[0090] e) uridine 5′-diphosphate galactose (UDP-Gal) to be present in anaqueous medium to produce and accumulate a reaction product in whichpoly-N-acetyllactosamine sugar chain is added to the non-reducingterminal of the acceptor in the aqueous medium, and recovering thepoly-N-acetyllactosamine sugar chain-added sugar chain or complexcarbohydrate from the aqueous medium.

[0091] (37) A process for producing a sugar chain or complexcarbohydrate, which comprises using the transformant according to anyone of (22) to (27) to produce and accumulate a sugar chain comprising asaccharide selected from the group consisting ofGlcNAcβ1-3Galβ1-4Glc-ceramide, a lacto-series glycolipid (a glycolipidhaving Galβ1-3GlcNAcβ1-3Galβ1-4Glc-ceramide as a backbone), aneolacto-series glycolipid (a glycolipid havingGalβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide as a backbone), a saccharide havingGlcNAcβ1-3Gal structure, a saccharide having GlcNAcβ1-3Galβ1-4GlcNAcstructure, a saccharide having GlcNAcβ1-3Galβ1-3GlcNAc structure, asaccharide having GlcNAcβ1-3Galβ1-4Glc structure, a saccharide having(Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc structure wherein n is 1 or moreand a saccharide having a (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structurewherein n is 1 or more, or a complex carbohydrate containing the sugarchain, and recovering the sugar chain or complex carbohydrate from theculture.

[0092] (38) A process for producing a sugar chain or complexcarbohydrate, which comprises using the non-human transgenic animal ortransgenic plant according to (28) to produce and accumulate a sugarchain comprising a saccharide selected from the group consisting ofGlcNAcβ1-3Galβ1-4Glc-ceramide, a lacto-series glycolipid (a glycolipidhaving Galβ1-3GlcNAcβ1-3Galβ1-4Glc-ceramide as a backbone), aneolacto-series glycolipid (a glycolipid havingGalβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide as a backbone), a saccharide havingGlcNAcβ1-3Gal structure, a saccharide having GlcNAcβ1-3Galβ1-4GlcNAcstructure, a saccharide having GlcNAcβ1-3Galβ1-3GlcNAc structure, asaccharide having GlcNAcβ1-3Galβ1-4Glc structure, a saccharide having(Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc structure wherein n is 1 or moreand a saccharide having (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structurewherein n is 1 or more, or a complex carbohydrate containing the sugarchain, and recovering the sugar chain or complex carbohydrate from theindividual.

[0093] (39) The process according to any one of (34) to (38), whereinthe complex carbohydrate is selected from a glycoprotein, a glycolipid,a proteoglycan, a glycopeptide, a lipopolysaccharide, a peptidoglycanand a glycoside in which a sugar chain is linked to a steroid compound.

[0094] (40) The process according to (38), wherein the accumulation iscarried out in an animal milk.

[0095] (41) An oligonucleotide which has a sequence identical tocontinuos 5 to 120 nucleotides of the nucleotide sequence in the DNAaccording to any one of (10) to (19), or which is a derivative of theoligonucleotide.

[0096] (42) A method for determining expression level of a gene encodingthe polypeptide according to any one of (1) to (8), which comprisesusing the DNA according to any one of (10) to (19), a partial fragmentof the DNA or the oligonucleotide according to (41) by a hybridizationmethod.

[0097] (43) A method for determining expression level of a gene encodingthe polypeptide according to any one of (1) to (8), which comprisesusing the oligonucleotide according to (41) by a polymerase chainreaction.

[0098] (44) A method for detecting an inflammation, cancer or tumormetastasis, which comprises using the method according to (42) or (43).

[0099] (45) An agent for detecting an inflammation, cancer or tumormetastasis, which comprises the DNA according to any one of (10) to(19), a partial fragment of the DNA or the oligonucleotide according to(41).

[0100] (46) A method for diagnosing functional abnormality of a gene,which comprises detecting mutation of the gene which encodes thepolypeptide according to any one of (1) to (8).

[0101] (47) A method for detecting mutation of a gene encoding thepolypeptide according to any one of (1) to (8), which comprises usingthe DNA according to any one of (10) to (19), a partial fragment of theDNA or the oligonucleotide according to (41) by a hybridization method.

[0102] (48) A method for detecting mutation of a gene encoding thepolypeptide according to any one of (1) to (8), which comprises usingthe oligonucleotide according to (41) by a polymerase chain reaction.

[0103] (49) A method for inhibiting transcription of a gene encoding thepolypeptide according to any one of (1) to (8) or translation of mRNAthereof, which comprises using the oligonucleotide according to (41).

[0104] (50) An antibody which recognizes the polypeptide according toany one of (1) to (8).

[0105] (51) A method for immunologically detecting the polypeptideaccording to any one of (1) to (8), which comprises using the antibodyaccording to (50).

[0106] (52) A method for immunohistostaining, which comprises detectingthe polypeptide according to any one of (1) to (8) by using the antibodyaccording to (50).

[0107] (53) An immunohistostaining agent, which comprises the antibodyaccording to (50).

[0108] (54) A medicament which comprises the polypeptide according toany one of (1) to (8).

[0109] (55) The medicament according to (54), which is a medicament fortreating, preventing and/or diagnosing an inflammatory disease, canceror tumor metastasis.

[0110] (56) A medicament which comprises the DNA according to any one of(10) to (19), a partial fragment of the DNA or the oligonucleotideaccording to (41).

[0111] (57) The medicament according to (56), which is a medicament fortreating, preventing and/or diagnosing an inflammatory disease, canceror tumor metastasis.

[0112] (58) A medicament which comprises the recombinant vectoraccording to (20) or (21).

[0113] (59) The medicament according to (58), which is a medicament fortreating, preventing and/or diagnosing an inflammatory disease, canceror tumor metastasis.

[0114] (60) A medicament which comprises the antibody according to (50).

[0115] (61) The medicament according to (60), which is a medicament fortreating, preventing and/or diagnosing an inflammatory disease, canceror tumor metastasis.

[0116] (62) A method for screening a compound which changes aβ1,3-N-acetylglucosaminyltransferase activity possessed by thepolypeptide according to any one of (1) to (8), which measuring changesin the β1,3-N-acetylglucosaminyltransferase activity of the polypeptidecaused by a sample to be tested by allowing the polypeptide to contactwith the sample to be tested.

[0117] (63) The method according to (62), wherein theβ1,3-N-acetylglucosaminyltransferase activity is an activity to transferN-acetylglucosamine via β1,3-linkage to a galactose residue present inthe non-reducing terminal of a sugar chain.

[0118] (64) The method according to (62) or (63), wherein theβ1,3-N-acetylglucosaminyltransferase activity is an activity to transferN-acetylglucosamine via β1,3-linkage to a galactose residue present inits non-reducing terminal of a sugar chain of an acceptor selected fromi) galactose, N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc orlactose (Galβ1-4Glc), ii) an oligosaccharide having galactose,N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure in thenon-reducing terminal, and iii) a complex carbohydrate having agalactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure inthe non-reducing terminal.

[0119] (65) The method according to (62) or (63), wherein the complexcarbohydrate having galactose, N-acetyllactosamine, Galβ1-3GlcNAc orlactose structure in the non-reducing terminal is lactosylceramide orparagloboside.

[0120] (66) The method according to (64) or (65), wherein the complexcarbohydrate is a complex carbohydrate selected from a glycoprotein, aglycolipid, a proteoglycan, a glycopeptide, a lipopolysaccharide, apeptidoglycan and a glycoside in which a sugar chain is linked to asteroid compound.

[0121] (67) A method for screening a compound which changes expressionof a gene encoding the polypeptide according to any one of (1) to (8),which comprises allowing the polypeptide to contact with the sample tobe tested, and determining paragloboside or poly-N-acetylglucosaminesugar chain by using at least one selected from the group consisting ofan antibody which recognizes paragloboside, and an antibody or lectinwhich recognizes poly-N-acetylglucosamine sugar chain.

[0122] (68) A method for screening a compound which changes expressionof a gene encoding the polypeptide according to any one of (1) to (8),which comprises allowing a cell expressing the polypeptide to contactwith a sample to be tested, and determining the polypeptide by using theantibody according to (50).

[0123] (69) A promoter DNA which controls transcription of a geneencoding the polypeptide according to any one of (1) to (8).

[0124] (70) The promoter DNA according to (69), which is a promoterfunctioning in a cell selected from a leukocyte cell, a nerve cell, atracheal cell, a lung cell, a colon cell, a placental cell, aneuroblastoma cell, a glioblastoma cell, a colon cancer cell, a lungcancer cell, a pancreatic cancer cell, a stomach cancer cell and aleukemia cell.

[0125] (71) The promoter DNA according to (69) or (70), which is ahuman-, rat- or mouse-derived promoter DNA.

[0126] (72) A method for screening a compound which changes efficiencyof transcription by the promoter DNA according to any one of (69) to(71), which comprises transforming an animal cell by using a plasmidcontaining the promoter DNA and a reporter gene ligated to thedownstream of the promoter DNA, allowing the transformant to contactwith a sample to be tested, and determining the translated product ofthe reporter gene.

[0127] (73) The method according to (72), wherein the reporter gene is agene selected from a chloramphenicol acetyltransferase gene, aP-galactosidase gene, a β-lactamase gene, a luciferase gene and a greenfluorescent protein gene.

[0128] (74) A compound obtainable by the method according to any one of(62) to (68), (72) and (73).

[0129] (75) A non-human knockout animal in which a deficiency ormutation is introduced into a DNA encoding the polypeptide according to(1) to (8).

[0130] (76) The knockout animal according to (75), wherein the non-humanknockout animal is a mouse.

[0131] (77) A method for controlling differentiation, mutual recognitionand migration of a cell, which comprises introducing the DNA accordingto any one of (10) to (18), an RNA comprising a sequence homologous tothe DNA or the recombinant vector according to (20) or (21) into a cellto express the polypeptide according to any one of (1) to (8).

[0132] (78) The method according to (77), wherein the cell is a cellselected from any one of a blood cell, a nerve cell, a stem cell or acancer cell.

[0133] (79) A method for accelerating differentiation of a promyelocyteinto a granulocyte, which comprises introducing the DNA according to anyone of (10) to (18), an RNA comprising a sequence homologous to the DNAor the recombinant vector according to (20) or (21) into a promyelocyteto express the polypeptide according to any one of (1) to (8).

[0134] The present invention is described below in detail.

(1) Preparation of a DNA Encoding the Polypeptide of the PresentInvention and Production of the DNA and the Oligonucleotide

[0135] The GlcNAc β1,3-galactosyltransferase disclosed in JapanesePublished Unexamined Patent Application No. 181752/94 (hereinafterreferred to as “β3Gal-T1”; alias WM1) is a GlcNAcβ1,3-galactosyltransferase involved in the synthesis of Galβ1-3GlcNAcstructure. Information on a DNA encoding the polypeptide of the presentinvention or nucleotide sequence of a part of the DNA can be obtained byretrieving genes having homology with this enzyme gene or genes having apossibility to encode proteins having homology with the enzyme at aminoacid level from a gene data base using programs such as BLAST [J. Mol.Biol., 215, 403-410 (1990)], FASTA (Methods in Enzymology, 183, 63-69)and FrameSearch (manufactured by Compugen). As the data bases, publicdata bases such as GenBank, EMBL and Geneseq (Derwent Publications) canbe used, or personal data bases can also be used. These approachesreveled that the rat cDNA nucleotide sequence represented by SEQ ID NO:3can be exemplified as nucleotide sequence of a gene having a possibilityto encode a protein having homology with the β3Gal-T1 gene at amino acidlevel. Also, the human EST sequence of GenBank No. AI039637 can beexemplified as a human cDNA nucleotide sequence having homology with thenucleotide sequence of SEQ ID NO:3. These sequences are partialnucleotide sequences of DNAs encoding the polypeptides of the presentinvention.

[0136] The presence of the DNAs encoding the polypeptides of the presentinvention can be detected by carrying out polymerase chain reaction(hereinafter referred to as “PCR”) [Molecular Cloning, A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press (1989)(hereinafter referred to as “Molecular Cloning, Second Edition”) and PCRProtocols, Academic Press (1990)] using a single-stranded cDNAs or cDNAlibraries prepared from various tissues or various cells as templatesand primers specific for the above sequence. Also, a DNA fragment of theDNA encoding the polypeptide of the present invention can be obtained.

[0137] When the resulting DNA fragment is not a full-length, afull-length cDNA can be obtained as follows.

[0138] A tissue- or cell-derived cDNA library in which the presence ofthe DNA has been confirmed is screened by using the above obtained DNAfragment as a probe to thereby obtain a full-length cDNA.

[0139] Also, the 5′ RACE method or the 3′ RACE method is carried out byusing a single-stranded cDNA or cDNA library in which the presence ofthe DNA has been confirmed as a template to thereby obtain 5′-terminalfragment and 3′-terminal fragment of cDNA having the sequence. Afull-length cDNA can be obtained by ligating both fragments.

[0140] A single-stranded cDNA derived from various tissues or variouscells can be prepared by a known method or a commercially available kit.An example is shown below.

[0141] A total RNA is prepared from various tissues or various cellsaccording to a guanidine thiocyanate phenol-chloroform method [Anal.Biochem., 162, 156-159 (1987)]. If necessary, a chromosomal DNA whichmay be contaminated is degradated by treatment of the total RNA withdeoxyribonuclease I (manufactured by Life Technologies). Asingle-stranded DNA is synthesized from each of the resulting total RNAby using an oligo(dT)-primer or a random primer according toSUPERSCRIPT™ Preamplifiation System for First Strand cDNA System(manufactured by Life Technologies). The single-stranded cDNAs includesingle-stranded cDNAs prepared from human colon cancer cell line colo205or human gastric mucosa according to the above method.

[0142] A cDNA library can be produced by a known method. The cDNAlibrary construction method include methods described in MolecularCloning, Second Edition, Current Protocols in Molecular Biology, JohnWiley & Sons (1987-1997) (hereinafter referred to as “Current Protocolsin Molecular Biology”), Supplements 1-38 and the like; methods using acommercially available kit such as SuperScript Plasmid System for cDNASynthesis and Plasmid Cloning (manufactured by GIBCO BRL) and ZAP-cDNASynthesis Kit (manufactured by STRATAGENE); and the like. A cDNA libraryderived from various tissues or various cells can be obtained bypurchasing commercially available one.

[0143] Any of phage vectors, plasmid vectors and the like can be used asa cloning vector for constructing a cDNA library, so long as it canautonomously replicate in Escherichia coli K12. Examples include ZAPExpress [manufactured by STRATAGENE, Strategies, 5, 58 (1992)],pBluescript SK(−) and pBluescript II SK(+) [Nucleic Acids Research, 17,9494 (1989)], λZAP II (manufactured by STRATAGENE), λgt10 and λgt11 [DNACloning, A Practical Approach, 1, 49 (1985)], λTriplEx (manufactured byClontech), λExCell (manufactured by Pharmacia), pT7T318U (manufacturedby Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)], pUC18 [Gene, 33,103 (1985)], pAMo [J. Biol. Chem., 268, 22782-22787 (1993), aliaspAMoPRC3Sc (Japanese Published Unexamined Patent Application No.336963/93)], pGAD10 [Gene, 10, 193 (1991)) and the like.

[0144] Any microorganism can be used as a host microorganism, so long asit belongs to Escherichia coli. Examples include Escherichia coliXL1-Blue MRF′ [manufactured by STRATAGENE, Strategies, 5, 81 (1992)],Escherichia coli C600 [Genetics, 39, 440 (1954)], Escherichia coli Y1088[Science, 222, 778 (1983)], Escherichia coli Y1090 [Science, 222, 778(1983)], Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)],Escherichia coli K802 [J. Mol. Biol., 16, 118 (1966)], Escherichia coliJM105 [Gene, 38, 275 (1985)], Escherichia coli SOLR™ Strain(manufactured by STRATAGENE), Escherichia coli LE392 (Molecular Cloning,Second Edition) and the like.

[0145] As a cDNA library, a cDNA library produced as follows isexemplified.

[0146] A cDNA is synthesized from a poly(A)⁺ RNA of human gastric mucosaby using a cDNA synthesis system (cDNA Synthesis System, manufactured byGIBCO BRL), EcoRI-NotI-SalI adapter (Super Choice System for cDNASynthesis; manufactured by GIBCO BRL) is added to both ends thereof, thecDNA is inserted into an EcoRI site of a cloning vector λZAP II (λZAPII/EcoRI/CIAP Cloning Kit, manufactured by STRATAGENE), and in vitropackaging is carried out by using Gigapack III Gold Packaging Extract(manufactured by STRATAGENE) to thereby produce a cDNA library.

[0147] Also, a commercially available cDNA library can be used bypurchasing it.

[0148] Based on the nucleotide sequence of a candidate gene found by thedata base search, primers specific for the gene are designed and PCR iscarried out using the thus obtained single-stranded cDNAs or cDNAlibraries as templates. When an amplified fragment is obtained, thefragment is subcloned into an appropriate plasmid. The subcloning can becarried out by inserting the amplified DNA fragment directly, or aftertreatment with restriction enzymes or DNA polymerase, into a vector inthe usual way. Examples of the vectors include pBluescript SK(−) andpBluescript II SK(+) (both manufactured by STRATAGENE), pDIRECT [NucleicAcids Research, 18, 6069 (1990)], pCR-Amp SK(+) [manufactured bySTRATAGENE, Strategies, 5, 6264 (1992)], pT7Blue (manufactured byNovagen), pCR II [manufactured by Invitrogen; Biotechnology, 9, 657(1991)], pCR-TRAP (manufactured by Genehunter), pNoTA_(T7) (manufacturedby 5′→3′) and the like.

[0149] Whether or not the objective DNA fragment can be obtained isconfirmed by sequencing the subcloned PCR amplified fragment. Thenucleotide sequence can be determined by a generally used nucleotidesequence analyzing method such as the dideoxy method of Sanger et al.[Proc. Natl. Acad. Sci. USA, 74, 5463 (1997)] or using a nucleotidesequence analyzing apparatus such as 373A DNA sequencer (manufactured byPerkin Elmer).

[0150] A cDNA having a possibility to encode a protein homologous to theβ3Gal-T1 at amino acid level can be obtained by colony hybridization orplaque hybridization (Molecular Cloning, Second Edition) for the cDNAlibraries prepared in the above using the DNA fragment as a probe. As aprobe, the DNA fragment labeled with an isotope or digoxigenin can beused.

[0151] The nucleotide sequence of the DNA obtained by the above methodcan be determined by inserting the DNA fragment as such or after itsdigestion with appropriate restriction enzymes or the like into a vectorby a general method described in Molecular Cloning, 2nd Ed. or the like,and then analyzing it by a generally used nucleotide sequence analyzingmethod such as the dideoxy method of Sanger et al. [Proc. Natl. Acad.Sci. USA, 74, 5463 (1997)] or using a nucleotide sequence analyzingapparatus such as DNA sequencer 373A (manufactured by Perkin Elmer), DNAsequencer 377 (manufactured by Perkin Elmer) or DNA sequencer model4000L (manufactured by LI-COR).

[0152] The DNAS obtained by this method include a DNA encoding apolypeptide which comprises the amino acid sequence represented by SEQID NO:1 or a polypeptide which comprises an amino acid sequence ofpositions 39 to 378 in the amino acid sequence represented by SEQ IDNO:1, and the like. Specific examples include a DNA which comprises thenucleotide sequence represented by SEQ ID NO:2, a DNA which comprises anucleotide sequence of positions 135 to 1268 in the nucleotide sequencerepresented by SEQ ID NO:2, a DNA which comprises a nucleotide sequenceof positions 249 to 1268 in the nucleotide sequence represented by SEQID NO:2, and the like.

[0153] Also, the nucleotide sequence represented by SEQ ID NO:2 is anucleotide sequence derived from a cDNA encoding a polypeptidecomprising the amino acid sequence represented by SEQ ID NO:1, thenucleotide sequence of positions 135 to 1268 in the nucleotide sequencerepresented by SEQ ID NO:2 is a nucleotide sequence corresponding to acoding region for the polypeptide, and the nucleotide sequence ofpositions 249 to 1268 in the nucleotide sequence represented by SEQ IDNO:2 is a nucleotide sequence encoding a region having aglycosyltransferase activity of the polypeptide.

[0154] Examples of plasmids comprising the DNA having the nucleotidesequence of positions 135 to 1268 in the nucleotide sequence representedby SEQ ID NO:2 include pAMo-G4, pCXN2-G6 and pBS-G4.

[0155] Generally, since plural codons are present for one amino acid, aDNA comprising a nucleotide sequence different from SEQ ID NO:2 isincluded in the DNA of the present invention, so long as it encodes thepolypeptide of the present invention.

[0156] Based on the information of the nucleotide sequence in the DNAsof the present invention obtained by the above method, using a DNAcomprising a 5′-terminal 15-30 bp sequence of the total nucleotidesequence of the DNA of the present invention or any region thereof, anda DNA comprising 3′-terminal 15-30 bp complementary sequence thereof asa sense primer and an antisense primer, respectively, PCR is carried outusing cDNAs prepared from mRNAs of a cell expressing mRNAs complementaryto the DNAs as templates to thereby prepare the DNAs of the presentinvention and the fragments of any region thereof. The DNAs used asprimers can be synthesized by a DNA synthesizer such as 380A, 392 and3900 manufactured by Applied Biosystems.

[0157] Furthermore, based on the amino acid sequence of the polypeptideencoded by the DNA of the present invention, the DNA of the presentinvention can be prepared by chemically synthesizing a DNA encoding thepolypeptide. The DNA can be chemically synthesized by using a DNAsynthesizer manufactured by Shimadzu Corporation according to thethiophosphate method, DNA synthesizers 380A, 392 and 3900 manufacturedby Applied Biosystem according to the phosphoramidite method, and thelike.

[0158] The objective DNAs encoding a polypeptide comprising an aminoacid sequence in which at least one amino acid is deleted, substitutedor added in the amino acid sequence represented by SEQ ID NO:1 can beobtained by selecting a DNA which hybridizes with a DNA comprising asequence complementary to the nucleotide sequence of the DNA obtained bythe above method under stringent conditions. For example, a homologueDNA and the like of other species (mouse, rat, calf, monkey or the like)can be cloned. Specifically, a rat homologue DNA represented by SEQ IDNO:3 is exemplified.

[0159] Except for the synthesis method using a DNA synthesizer, the DNAsencoding the polypeptide of the present invention are obtained asdouble-stranded DNAs consisting of a sense DNA encoding the polypeptideof the present invention and a DNA comprising a sequence complementaryto the nucleotide sequence of the DNA. Both the DNAs can be separated byheating at 100° C. for 5 minutes, followed by rapid cooling on ice. TheDNA comprising a sequence complementary to the nucleotide sequence ofthe DNA encoding the polypeptide of the present invention can bysynthesized by a DNA polymerase reaction in the presence of dNTP usingthe above separated sense DNA encoding the polypeptide of the presentinvention as a template and a DNA comprising a sequence complementary to3′-terminal 5-30 bp sequence of the sense DNA as a primer.

[0160] A DNA which is hybridizable under stringent conditions is a DNAobtained by carrying out colony hybridization, plaque hybridization,Southern hybridization or the like using the DNA obtained in the aboveas a probe. Examples include a DNA which can be identified byhybridization at 65° C. in the presence of 0.7 to 1.0 M sodium chlorideusing a filter to which colony- or plaque-derived DNA samples areimmobilized and then washing the filter at 65° C. with 0.1 to 2 timesconcentration of SSC solution (composition of the original concentrationSSC containing 150 mmol/l sodium chloride and 15 mmol/l sodium citrate).

[0161] The hybridization can be carried out according to the methoddescribed in Molecular Cloning, Second Edition, Current Protocols inMolecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach,Second Edition., Oxford University (1995) or the like. The hybridizableDNA includes a DNA having a homology of 60% or more, preferably 70% ormore, more preferably 80% or more, still more preferably 90% or more,particularly preferably 95% or more, and most preferably 97% or more,with the DNA obtained above, when calculated using a default (initialestablishment) parameter with an analysis software such as BLAST [J.Mol. Biol., 215, 403 (1990)) or FASTA [Methods in Enzymology, 183, 63-98(1990)].

[0162] Oligonucleotides, such as a sense oligonucleotide having apartial sequence of the DNA of the present invention and an antisenseoligonucleotide having a partial sequence of a nucleotide sequencecomplementary to the nucleotide sequence of the DNA of the presentinvention can be prepared using the DNA and DNA fragments of the presentinvention obtained by the above method, in a conventional mannerdescribed in Molecular Cloning, Second Edition or the like or using aDNA synthesizer based on the nucleotide sequence information of the DNA.

[0163] The oligonucleotide includes a DNA comprising a sequenceidentical to continuous 5 to 120 nucleotides, preferably continuos 5 to60 nucleotides, in the nucleotide sequence in the DNA of the presentinvention or a nucleotide sequence complementary to the nucleotidesequence. Examples include a DNA comprising a sequence identical tocontinuous 5 to 120 nucleotides in the nucleotide sequence representedby SEQ ID NO:2 or a DNA comprising the same sequence as continuos 5 to120 nucleotides in a sequence complementary to the nucleotide sequencerepresented by SEQ ID NO:2. When used as a forward primer and a reverseprimer, the above oligonucleotides in which the melting temperatures(Tm) and the number of nucleotides therebetween are not significantlydifferent are preferred. Examples include oligonucleotides comprisingthe nucleotide sequence represented by SEQ ID NO:25 or 28 and the like.

[0164] Moreover, derivatives of these oligonucleotides (hereinafterreferred to as “oligonucleotide derivatives”) can also be used as theoligonucleotides of the present invention.

[0165] Examples of the oligonucleotide derivatives includeoligonucleotide derivatives in which a phosphodiester bond in theoligonucleotide is converted into a phosphorothioate bond,oligonucleotides derivative in which a phosphodiester bond in theoligonucleotide is converted into an N3′-P5′ phosphoamidate bond,oligonucleotide derivatives in which ribose and a phosphodiester bond inthe oligonucleotide are converted into a peptide-nucleic acid bond,oligonucleotide derivatives in which uracil in the oligonucleotide issubstituted with C-5 propynyluracil, oligonucleotide derivatives inwhich uracil in the oligonucleotide is substituted with C-5thiazoleuracil, oligonucleotide derivatives in which cytosine in theoligonucleotide is substituted with C-5 propynylcytosine,oligonucleotide derivatives in which cytosine in the oligonucleotide issubstituted with phenoxazine-modified cytosine, oligonucleotidederivatives in which ribose in the oligonucleotide is substituted with2′-O-propylribose, oligonucleotide derivatives in which ribose in theoligonucleotide is substituted with 2′-methoxyethoxyribose, and the like[Cell Technology, 16, 1463 (1997)].

[0166] The polypeptide of the present invention encoded by the thusobtained DNA of the present invention includes a polypeptide whichcomprises the amino acid sequence. represented by SEQ ID NO:1; apolypeptide which comprises an amino acid sequence of positions 39 to378 in the amino acid sequence represented by SEQ ID NO:1; a polypeptidewhich comprises an amino acid sequence in which at least one amino acidin the amino acid sequence in the above polypeptide is deleted,substituted or added, and has a β1,3-N-acetylglucosaminyltransferaseactivity; a polypeptide which comprises an amino acid sequence having60% or more of homology with the amino acid sequence in the abovepolypeptide, and has a lactosylceramideβ1,3-N-acetylglucosaminyltransferase activity and a paraglobosideβ1,3-N-acetylglucosaminyltransferase activity; and the like.

[0167] A protein comprising an amino acid sequence in which at least oneamino acid is deleted, substituted and/or added in the polypeptidehaving the above amino acid sequence can be obtained, for example, byintroducing site-directed mutation into the DNA encoding the polypeptidecomprising the amino acid sequence represented by SEQ ID NO:1 accordingto the site-directed mutagenesis method described in Molecular Cloning,A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory(1989) (hereinafter referred to as “Molecular Cloning, Second Edition”),Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997)(hereinafter referred to as “Current Protocols in Molecular Biology”),Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79,6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431(1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985) or the like. Thenumber of amino acids which have been deleted, substituted or added isnot particularly limited; however, they are 1 to tens, preferably 1 to20, more preferably 1 to 10 and most preferably 1 to 5 amino acids.

[0168] Also, the polypeptide of the present invention includes an aminoacid sequence having a homology of 60% or more with the amino acidsequence represented by SEQ ID NO:1. The homology with the amino acidsequence represented by SEQ ID NO:1 is 60% or more, preferably 70% ormore, more preferably 80% or more, still more preferably 90% or more,particularly preferably 95% or more, and most preferably 97% or more,when calculated using a default (initial establishment) parameter withan analysis software such as BLAST [J. Mol. Biol., 215, 403 (1990)] orFASTA [Methods in Enzymology, 183, 63-69 (1990)]. For example, ahomologue polypeptide of other species (mouse, rat, calf, monkey or thelike) is exemplified. Specifically, the rat polypeptide represented bySEQ ID NO:3 is exemplified.

[0169] According to the method described in the following (3), it can beconfirmed that the polypeptide of the present invention has aβ1,3-N-acetylglucosaminyltransferase activity.

(2) Production of the Polypeptide of the Present Invention

[0170] In order to express the DNA of the present invention obtained bythe above method in a host and produce the polypeptide of the presentinvention, methods described in Molecular Cloning, Second Edition,Current Protocols in Molecular Biology, Supplements 1 to 38 and the likecan be used.

[0171] Specifically, a recombinant vector to which the DNA of thepresent invention has been inserted into the downstream of the promoterof a suitable expression vector is constructed, a transformantexpressing the polypeptide of the present invention is obtained byintroducing the vector into a host cell, and the transformant iscultured to produce the polypeptide of the present invention.

[0172] Any of bacteria, yeast, animal cells, insect cells, plant cellsand the like can be used as the host cell, so long as it can express theobjective gene.

[0173] The expression vector includes those which can autonomouslyreplicate in the above host cell or which can be integrated into achromosome and have a promoter at such an operative position that theDNA of the present invention can be transcribed.

[0174] When prokaryote, such as a bacterium, is used as a host cell, itis preferred that the expression vector for the polypeptide gene of thepresent invention can autonomously replicate in the prokaryote and is arecombinant vector constructed with a promoter, a ribosome bindingsequence, the DNA of the present invention and a transcriptiontermination sequence. A promoter-controlling gene can also be utilized.

[0175] The expression vector includes pBTrp2, pBTac1 and pBTac2 (allavailable from Boehringer Mannheim), pSE280 (manufactured byInvitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured byQIAGEN), pKYP10 (Japanese Published Unexamined Patent Application No.110600/83), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric.Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82,4306 (1985)], pBlue II SK(−) (manufactured by STRATAGENE), pTrs30 (FERMBP-5407), pTrs32 (FERM BP-5408), pGHA2 (FERM BP-400), pGKA2 (FERMB-6798), pTerm2 (Japanese Published Unexamined Patent Application No.22979/91, U.S. Pat. No. 4,686,191, U.S. Pat. No. 4,939,094, U.S. Pat.No. 5,160,735), pKK233-2 (manufactured by Pharmacia), PGEX (manufacturedby Pharmacia), pET (manufactured by Novagen), psupex, pUB110, pTP5,pC194, pTrxFus (manufactured by Invitrogen), pMAL-c2 (manufactured byNew England Biolabs) and the like.

[0176] Any promoter can be used, so long as it can work for expressionin a host cell such as Escherichia coli. Examples include promotersderived from Escherichia coli, phage and the like, such as trp promoter(Ptrp), lac promoter (Plac), P_(L) promoter and P_(R) promoter, SPO1promoter, SPO2 promoter and penP promoter. Also, artificially designedand modified promoters, such as a promoter in which two Ptrp are linkedin series (Ptrpx2), tac promoter, lacT7 promoter and leti promoter, canbe used.

[0177] As the ribosome binding sequence, it is preferred to use aplasmid in which the space between Shine-Dalgarno sequence and theinitiation codon is adjusted to a suitable distance (for example, 6 to18 nucleotides).

[0178] The transcription termination sequence is not required for theexpression of the DNA of the present invention. However, thetranscription terminating sequence is preferably arranged at justdownstream of the structural gene.

[0179] The host cell includes microorganisms belonging to the genusEscherichia, the genus Serratia, the genus Bacillus, the genusBrevibacterium, the genus Corynebacterium, the genus Microbacterium, thegenus Pseudomonas and the like, such as Escherichia coli XL1-Blue,Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coliMC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichiacoli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichiacoli W3110, Escherichia coli NY49, Escherichia coli BL21(DE3),Escherichia coli BL21(DE3)pLysS, Escherichia coli HMS174(DE3),Escherichia coli HMS174(DE3)pLysS, Serratia ficaria, Serratia fonticola,Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillusamyloliquefaciens, Brevibacterium ammoniagenes, Brevibacteriumimmariophilum ATCC 14068, Brevibacterium saccharolyticum ATCC 14066,Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC 13869, Corynebacteriumacetoacidophilum ATCC 13870, Microbacterium ammoniaphilum ATCC 15354 andPseudomonas sp. D-0110.

[0180] Any method can be used in the method for introducing therecombinant vector, so long as it is a method for introducing a DNA intothe above host cell, such as an electroporation method [Nucleic AcidsRes., 16, 6127 (1988)], a method using a calcium ion [Proc. Natl. Acad.Sci. USA, 69, 2110 (1972)], a protoplast method (Japanese PublishedUnexamined Patent Application No. 248394/88) and methods described inGene, 17, 107 (1982) and Molecular & General Genetics, 168, 111 (1979).

[0181] When yeast is used as the host cell, the expression vectorincludes YEp13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419),pHS19, pHS15 and the like.

[0182] Any promoter can be used, so long as it can be expressed inyeast. Examples include PHO5 promoter, PGK promoter, GAP promoter, ADHpromoter, gal 1 promoter, gal 10 promoter, a heat shock polypeptidepromoter, MFα1 promoter, CUP 1 promoter, and the like.

[0183] Examples of the host cell include yeast strains belonging to thegenus Saccharomyces, the genus Schizosaccharomyces, the genusKiuyveromyces, the genus Trichosporon, the genus Schwanniomyces and thelike, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe,Kluyveromyces lactis, Trichosporon pullulans and Schwanniomycesalluvius.

[0184] Any method can be used as the method for introducing therecombinant vector, so long as it is a method for introducing a DNA intoyeast. Examples include an electroporation method [Methods. Enzymol.,194, 182 (1990)], a spheroplast method [Proc. Natl. Acad. Sci. USA, 84,1929 (1978)], a lithium acetate method [J. Bacteriol., 153, 163 (1983)],the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) andthe like.

[0185] When an animal cell is used as the host, the expression vectorincludes pcDNAI/Amp (manufactured by Invitrogen), pcDNAI (manufacturedby Funakoshi), pcDM8 [Nature, 329, 840 (1987), manufactured byFunakoshi], pAGE107 [Japanese Published Unexamined Patent ApplicationNo. 22979/91; Cytotechnology, 3, 133 (1990)], pREP4 (manufactured byInvitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAMo [J. Biol.Chem., 268, 22782 (1993)], pAMoA [J. Biol. Chem., 268, 22782 (1993)],pAS3-3 (Japanese Published Unexamined Patent Application No. 227075/90)and the like.

[0186] Any promoter can be used as the method for introducing therecombinant vector, so long as it can work for expression in the animalcell. Examples include a promoter of IE (immediate early) gene ofcytomegalovirus (human CMV), an early promoter of SV40, a long terminalrepeat promoter of moloney murine leukemia virus, a promoter ofretrovirus, a heat shock promoter, SRα promoter, a promoter ofmetallothionein and the like. Also, the enhancer of the IE gene of humanCMV can be used together with the promoter.

[0187] As the host cell, any cell can be used, so long as it is ananimal cell into which a DNA can be introduced. For example, as cells ofmammals such as human, monkey, mouse, rat, guinea pig and mink can beused. Examples include mouse myeloma cell, rat myeloma cell, mousehybridoma cell, Chinese hamster CHO cell (ATCC CRL-9096, ATCC CCL-61),BHK cell, African green monkey kidney cell, human Namalwa cell (ATCCCRL-1432), human Namalwa KJM-1 cell, human fetal kidney cell, humanleukemic cell, HBT5637 (Japanese Published Unexamined Patent ApplicationNo. 299/88), human colon cancer cell line and the like.

[0188] The mouse myeloma cell includes SP2/0, NSO and the like. The ratmyeloma cell includes YB2/0 and the like. The human fetal kidney cellincludes BALL-1 and the like. The African green monkey kidney cellincludes COS-1 (ATCC CRL-1650), COS-7 (ATCC CRL-1651) and the like. Thehuman colon cancer cell line includes HCT-15 and the like.

[0189] When an object is to produce proteinous medicaments fortreatment, cells of mammals, particularly CHO cell, is preferably usedas the host.

[0190] Any method can be used as the method for introducing therecombinant vector into an animal cell, so long as it is a method forintroducing a DNA into an animal cell. Examples include anelectroporation method [Cytotechnology, 3, 133 (1990)], a calciumphosphate method (Japanese Published Unexamined Patent Application No.227075/90), a DEAE dextran method (Yodosha, Bio Manual Series 4, 16), alipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], amicroinjection method (Yodosha, Bio Manual Series 4, 36), an adenovirusmethod (Yodosha, Bio Manual Series 4, 43), a vaccinia virus method(Yodosha, Bio Manual Series 4, 59), a retrovirus method (Yodosha, BioManual Series 4, 74) and the like.

[0191] When an insect cell is used as the host, the polypeptide can beexpressed by a known method described in, for example, BacurovirusExpression Vectors, A Laboratory Manual, W.H. Freeman and Company, NewYork (1992), Molecular Biology, A Laboratory Manual, Current Protocolsin Molecular Biology, Supplements 1 to 38, Bio/Technology, 6, 47 (1988)or the like.

[0192] Specifically, a recombinant gene transfer vector and baculovirusare co-transfected into an insect cell to obtain a recombinant virus inan insect cell culture supernatant, and then the insect cell is infectedwith the resulting recombinant virus to express of the polypeptide.

[0193] The gene transfer vector used in the method includes pVL1392(manufactured by Pharmingen), pVL1393 (manufactured by Pharmingen),pBlueBacIII (manufactured by Invitrogen) and the like.

[0194] The bacurovirus includes Autographa californica nuclearpolyhedrosis virus which infects insects of the family Noctuidae, andthe like.

[0195] The insect cell includes Spodoptera frugiperda ovary cell,Trichoplusia ni ovary cell, Bombyx mori ovary-derived cultured cell andthe like. The Spodoptera frugiperda ovary cell includes Sf9 and Sf21(Bacurovirus Expression Vectors, A Laboratory Manual) and the like. TheTrichoplusia ni ovary cell includes High 5 (alias BTI-TN-5Bl-4,manufactured by Invitrogen) and the like. The Bombyx mori ovary-derivedcultured cell includes Bombyx mori N4 and the like.

[0196] The method for co-transfecting the above recombinant genetransfer vector and the above bacurovirus for the preparation of therecombinant virus include a calcium phosphate method (Japanese PublishedUnexamined Patent Application No. 227075/90), a lipofection method[Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like. Furthermore,the DNA can be introduced into an insect cell by using a method similarto the method for introducing a DNA into an animal cell. Examplesinclude an electroporation method [Cytotechnology, 3, 133 (1990)], acalcium phosphate method (Japanese Published Unexamined PatentApplication No. 227075/90), a lipofection method [Proc. Natl. Acad. Sci.USA, 84, 7413 (1987)] and the like.

[0197] When a plant cell or a plant is used as the host, the polypeptidecan be produced according to a known method [Tissue Culture, 20 (1994),Tissue Culture 21 (1995), Trends in Biotechnology, 15, 45 (1997)).

[0198] As the promoter used in the gene expression, any promoter can beused, so long as it can function in plant cells. Examples includecauliflower mosaic virus (CaMV) 35S promoter, rice actin 1 promoter andthe like. Also, the gene expression efficiency can be improved byinserting intron 1 of corn alcohol dehydrogenase gene or the likebetween the promoter and the gene to be expressed.

[0199] The host cell includes plant cells such as potato, tobacco, corn,rice, rape, soybean, tomato, wheat, barley, rye, alfalfa and flax. Asthe method for introducing a recombinant vector, any method forintroducing a DNA into a plant cell can be used. Examples include amethod using Agrobacterium (Japanese Published Unexamined PatentApplication No. 140885/84, Japanese Published Unexamined PatentApplication No. 70080/85, WO 94/00977), an electroporation method[Cytotechnology, 3, 133 (1990), Japanese Published Unexamined PatentApplication No. 251887/85], a method using a particle gun (gene gun)(Japanese Patent No. 2606856, Japanese Patent No. 2517813) and the like.

[0200] A cell or organ of the gene-introduced plant can be cultured in alarge amount using a jar fermentor. Also, a gene-introduced plant(transgenic plant) can be constructed by re-differentiating thegene-introduced plant cell.

[0201] The polypeptide of the present invention can also be producedusing an animal. For example, the polypeptide of the present inventioncan be produced in a gene-introduced animal according to known methods[American Journal of Clinical Nutrition, 63, 639S (1996), AmericanJournal of Clinical Nutrition, 63, 627S (1996), Bio/Technology, 9, 830(1991)].

[0202] Any promoter which can be expressed in an animal can be used and,for example, mammary gland cell-specific promoters such as α-caseinpromoter, β-lactoglobulin promoter and whey acidic protein promoter aresuitably used.

[0203] The polypeptide of the present invention can be produced byculturing a transformant derived from a microorganism, animal cell orplant cell having a recombinant vector into which DNA encoding thepolypeptide is inserted, according to a general culturing method, tothereby produce and accumulate the polypeptide, and then recovering thepolypeptide from the resulting culture mixture.

[0204] When the transformant is an animal or plant, the polypeptide canbe produced by breeding or cultivating it according to a generalbreeding or cultivating method to thereby produce and accumulate thepolypeptide, and then recovering the polypeptide from the animal orplant.

[0205] That is, in an animal, the polypeptide of the present inventioncan be obtained by, for example, breeding a non-human transgenic animalhaving the DNA of the present invention to produce and accumulate thepolypeptide of the present invention encoded by the recombinant DNA inthe animal, and then recovering the polypeptide from the animal.Examples of the production and accumulation part in the animal includemilk (Japanese Published Unexamined Patent Application No. 309192/86),eggs and the like.

[0206] In a plant, the polypeptide of the present invention can beobtained by, for example, cultivating a transgenic plant having the DNAof the present invention to produce and accumulate the polypeptide ofthe present invention encoded by the recombinant DNA in the plant, andthen recovering the polypeptide from the plant.

[0207] When the transformant for use in the production of thepolypeptide of the present invention is prokaryote such as Escherichiacoli or eukaryote such as yeast, the medium for culturing such anorganism may be either a natural medium or a synthetic medium, so longas it contains carbon sources, nitrogen sources, inorganic salts and thelike which can be assimilated by the organism and can efficientlyculture the transformant.

[0208] The carbon sources include those which can be assimilated by thetransformant. Examples include carbohydrates such as glucose, fructose,sucrose, molasses containing them, starch and starch hydrolysate;organic acids such as acetic acid and propionic acid; alcohols such asethanol and propanol; and the like.

[0209] The nitrogen sources includes ammonia, various ammonium salts ofinorganic acids and organic acids such as ammonium chloride, ammoniumsulfate, ammonium acetate and ammonium phosphate; othernitrogen-containing compounds, as well as peptone, meat extract, yeastextract, corn steep liquor, casein hydrolysate, soybean meal and soybeanmeal hydrolysate and various fermented cells and hydrolysates thereof.

[0210] The inorganic materials include potassium dihydrogen phosphate,dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate,sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate,calcium carbonate and the like.

[0211] Culturing is carried out under aerobic conditions such as shakingculture and submerged agitation aeration culture. The culturingtemperature is preferably from 15 to 40° C., and the culturing time isgenerally from 16 to 96 hours. During culturing, the pH is controlled at3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, analkali solution, urea, calcium carbonate, ammonia or the like.

[0212] If necessary, antibiotics such as ampicillin and tetracycline maybe added to the medium during culturing.

[0213] When a microorganism transformed with an expression vectorobtained using an inducible promoter as the promoter is cultured, aninducer may be added to the medium, if necessary. For example,isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be added tothe medium when a microorganism transformed with an expression vectorobtained using lac promoter is cultured, or indoleacrylic acid (IAA) orthe like may be added to the medium when a microorganism transformedwith an expression vector obtained using trp promoter is cultured.

[0214] When the transformant for the production of the polypeptide ofthe present invention is an animal cell, generally used RPMI 1640 medium[The Journal of the American Medical Association, 199, 519 (1967)],Eagle's MEM [Science, 122, 501 (1952)], DMEM [Virology, 8, 396 (1959)],199 Medium [Proceeding of the Society for the Biological Medicine, 73, 1(1950)] or any one of these media further supplemented with fetal calfserum or the like can be used.

[0215] Culturing is carried out generally at pH 6 to 8 and at 30 to 40°C. in the presence of 5% CO₂ for 1 to 7 days.

[0216] If necessary, antibiotics such as kanamycin and penicillin may beadded to the medium during culturing.

[0217] As the medium for use in culturing of a transformant obtainedusing an insect cell as the host, usually used TNM-FH medium(manufactured by PharMingen), Sf-900 II SFM medium (manufactured byGIBCO BRL), ExCell 400 or ExCell 405 (both manufactured by JRHBiosciences), Grace's Insect Medium [Nature, 195, 788 (1962)] or thelike can be used. Culturing is carried out at pH 6 to 7 and at 25 to 30°C. for 1 to 5 days. Also, if necessary, antibiotics such as gentamicinmay be added to the medium during culturing.

[0218] A transformant obtained using a plant cell as the host cell canbe used as the cell or after differentiating to a plant cell or organ.Examples of the medium used in culturing of the transformant includeMurashige and Skoog (MS) medium, White medium, media to which a planthormone such as auxin or cytokinine has been added, and the like.Culturing is carried out generally at a pH 5 to 9 and at 20 to 40° C.for 3 to 60 days. Also, if necessary, antibiotics such as kanamycin andhygromycin can be added to the medium during culturing.

[0219] Regarding the gene expression method, it can also be expressed asa partial polypeptide containing a region having aβ1,3-N-acetylglucosaminyltransferase activity, in addition to the caseof expressing a full-length polypeptide. In general, aglycosyltransferase has the topology of type II membrane protein andcomprises an N-terminal cytoplasmic tail region containing several toseveral dozen amino acids, a membrane-binding region having a highlyhydrophobic amino acid sequence, a stem region containing several toseveral dozen amino acids and the remaining most part of C-terminalmoiety containing a catalytic region. It is considered that the stemregion and the remaining most part of C-terminal moiety containing thecatalytic region are exposed to the Golgi body cavity. Boundary betweenthe stem region and catalytic region can be experimentally obtained bypreparing N-terminal-deleted polypeptides and examining degree of thedeletion by which the activity disappears. On the other hand, the stemregion and catalytic region can be estimated by comparing the amino acidsequence with that of similar glycosyltransferase having information onthe stem region and catalytic region.

[0220] It is expected that the polypeptide of the present inventionrepresented by SEQ ID NO:1 comprises an N-terminal cytoplasmic tailregion containing 14 amino acids, a subsequent membrane-binding regionrich in hydrophobic nature containing 18 amino acids, a stem regioncontaining at least 12 amino acids, and the remaining most part ofC-terminal moiety containing a catalytic region. Accordingly, apolypeptide comprising the amino acid sequence of positions 45 to 378 isconsidered to comprise a catalytic region.

[0221] The stem region was estimated based on the comparison of thehomology of the amino acid sequence with those of otherβ1,3-N-acetylglucosaminyltransferase and β1,3-galactosyltransferase andinformation on the stem regions of otherβ1,3-N-acetylglucosaminyltransferases and β1,3-galactosyltransferases.Specifically, the stem region can be estimated based on the informationdisclosed in Example 4 of this specification, and in Japanese PublishedUnexamined Patent Application No. 181759/94. For example, a secretedpolypeptide comprising amino acids of positions 36 to 378 in SEQ ID NO:1and a secreted polypeptide comprising amino acids of positions 39 to 378in SEQ ID NO:1 have β1,3-N-acetylglucosaminyltransferase activities.

[0222] In addition to its direct expression, the above full-lengthpolypeptide or partial polypeptides containing a region having aβ1,3-N-acetylglucosaminyltransferase activity (catalytic region) canalso be expressed as a secreted protein or a fusion protein according toa method described in Molecular Cloning, 2nd Ed. or the like. Examplesof proteins to be fused include β-galactosidase, protein A, IgG-bindingregion of protein A, chloramphenicol acetyltransferase, poly(Arg),poly(Glu), protein G, maltose-binding protein, glutathioneS-transferase, polyhistidine chain (His-tag), S peptide, DNA bindingprotein domain, Tac antigen, thioredoxin, green fluorescent protein,FLAG peptide, epitopes of antibodies of interest and the like [AkioYamakawa, Jikken Igaku, 13, 469-474 (1995)].

[0223] A process to produce the polypeptide of the present inventionincludes a process to produce it inside of a host cell, a process tosecrete it extracellularly from a host cell, and a process to produce iton the outermembrane of a host cell. The process is selected dependingon a host cell to use or the structure of the polypeptide to produce.

[0224] When the polypeptide of the present invention is produced insideof a host cell or on the outer membrane of a host cell, the polypeptidecan be positively secreted extracellularly from the host cell accordingto the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], themethod of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989),Genes Develop., 4, 1288 (1990)], or the methods described in JapanesePublished Unexamined Patent Application No. 336963/93, Wo 94/23021 andthe like.

[0225] That is, the polypeptide of the present invention can bepositively secreted extracellularly from a host cell by expressing it ina form in which a signal peptide is added to the upstream of apolypeptide containing active region of the polypeptide of the presentinvention, using gene recombination techniques.

[0226] Specifically, it is considered that the polypeptide of thepresent invention can be positively secreted extracellularly from a hostcell by adding a signal peptide to the upstream of a polypeptide havingan amino acid sequence presumably containing a catalytic region andexpressing the product. In addition, a tag for the purification anddetection can be added between the signal peptide and the catalyticregion or to the C-terminal of a polypeptide containing the catalyticregion.

[0227] Examples of the tag for the purification and detection includeβ-galactosidase, protein A, IgG-binding region of protein A,chloramphenicol acetyltransferase, poly(Arg), poly(Glu), protein G,maltose-binding protein, glutathione S-transferase, polyhistidine chain(His-tag), S peptide, DNA binding protein domain, Tac antigen,thioredoxin, green fluorescent protein, FLAG peptide, epitopes ofantibodies of interest and the like [Akio Yamakawa, Jikken Igaku, 13,469-474 (1995)].

[0228] Moreover, its production can be increased according to the methoddescribed in Japanese Published Unexamined Patent Application No.227075/90 using a gene amplification system in which a dihydrofolatereductase gene or the like is used.

[0229] As other methods for producing the polypeptide of the presentinvention, a production method by a in vitro transcription-translationsystem using the DNA of the present invention is exemplified. The invitro transcription-translation system means a system in which apolypeptide is produced by transcribing from DNA to mRNA and translatingfrom the mRNA to a protein using a cell-free system. Any system can beused, so long as it is a cell-free system in which an objectivepolypeptide can be produced from an objective DNA or an objective mRNA.Typical cell-free translation systems include a system using rabbitreticulocyte lysate or wheat germ lysate, and the like. The in vitrotranscription-translation system is commercially available as a kit fromvarious manufactures, and a polypeptide can be relatively easilyproduced by using the commercially available kits. The commerciallyavailable kits include In Vitro Express™ Translation Kit (manufacturedby STRATAGENE). Furthermore, the polypeptide of the present inventioncan also be produced by using an in vitro transcription-translationsystem according to the known method [J. Biomolecular NMR, 6, 129-134,Science, 242, 1162-1164, J. Biochem., 110, 166-168 (1991)].

[0230] General enzyme isolation purification methods can be used forisolating and purifying the polypeptides of the present invention from aculture of a transformant for producing the polypeptide of the presentinvention. For example, when the polypeptide of the present invention isaccumulated in a soluble state inside the cells of the transformant forproducing the polypeptide of the present invention, the cells in theculture are collected by centrifugation, the cells are washed and thenthe cells are disrupted using a sonicator, French press, Manton Gaulinhomogenizer, dynomill or the like to obtain a cell-free extract.

[0231] A purified product can be obtained from a supernatant prepared bycentrifuging the cell-free extract, by employing techniques, such assolvent extraction, salting out and desalting with ammonium sulfate orthe like, precipitation with organic solvents, anion exchangechromatography using a resin such as diethylaminoethyl (DEAE)-Sepharoseor DIAION HPA-75 (manufactured by Mitsubishi Chemical), cation exchangechromatography using a resin such as S-Sepharose FF (manufactured byPharmacia), hydrophobic chromatography using a resin such asbutyl-Sepharose or phenyl-Sepharose, gel filtration using a molecularsieve, affinity chromatography, chromatofocusing and electrophoresissuch as isoelectric focusing.

[0232] Also, when the polypeptide is expressed inside the cells in theform of an insoluble body, the cells are recovered, disrupted andcentrifuged in the same manner, the polypeptide is recovered from thethus obtained precipitated fraction in the usual way and then theinsoluble bodies of the polypeptide are solubilized using a proteindenaturing agent. The polypeptide is refolded into normalstereostructure by diluting or dialyzing the solubilized solution to oragainst a solution which does not contain the protein denaturing agentor contains the protein denaturing agent but in such a low concentrationthat the protein is not denatured, and then its purified product isobtained by the above isolation purification method.

[0233] When the polypeptide is secreted extracellularly, the culture istreated by centrifugation or the like means to obtain a solublefraction. A purified preparation of the polypeptide can be obtained fromthe soluble fraction by a method similar to the above method for itsisolation and purification from a cell-free extract supernatant.

[0234] Also, it can be purified according to the purification method ofgeneral glycosyltransferase [Methods in Enzymology, 83, 458].

[0235] Furthermore, the polypeptide of the present invention can bepurified by producing it as a fusion protein with other protein and thentreating the product with affinity chromatography in which a substancehaving affinity for the fused protein is used [Akio Yamakawa, JikkenIgaku, 13, 469-474 (1995)]. For example, the polypeptide of the presentinvention can be purified by producing it as a fusion protein withprotein A and then treating the fusion protein with affinitychromatography in which immunoglobulin G is used, according to themethod of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989),Genes Dev., 4, 1288 (1990)] or the method described in JapanesePublished Unexamined Patent Application No. 336963/93 or WO 94/23021.Also, the polypeptide of the present invention can be purified byproducing it as a fusion protein with FLAG peptide and then treating theproduct with an affinity chromatography in which an anti-FLAG antibodyis used [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4,1288 (1990)].

[0236] Furthermore, it can also be purified by affinity chromatographyin which an antibody for the polypeptide itself is used.

[0237] Moreover, the polypeptide of the present invention can also beproduced by a chemical synthesis method such as Fmoc method(fluorenylmethyloxycarbonyl method) or tBoc method (t-butyloxycarbonylmethod). Also, it can be chemically synthesized by use of a peptidesynthesizer manufactured, e.g., by Advanced ChemTech, PERKIN ELMER,Pharmacia Biotech, Protein Technology Instrument, Synthecell-Vega,PerSeptive, Shimadzu or the like.

[0238] The purified polypeptide of the present invention can bestructurally analyzed by a method generally used in protein chemistry,such as the method described in Protein Structure Analysis for GeneCloning (Idenshi Cloning No Tameno Tanpakushitsu Kozo Kaiseki) (editedby H. Hirano, published by Tokyo Kagaku Dojin, 1993).

[0239] (3) Activity Measurement and Application of the Polypeptide ofthe Present Invention

[0240] The β1,3-N-acetylglucosaminyltransferase activity of thepolypeptide of the present invention can be measured using a cellextract prepared from a transformant carrying an expression vector forthe polypeptide of the present invention or the polypeptide of thepresent invention isolated and purified from the transformant or acultured product thereof as the enzyme sample, based on a knownmeasuring method [J. Biol. Chem., 268, 27118 (1993), J. Biol. Chem.,267, 23507 (1992), J. Biol. Chem., 267, 2994 (1992), J. Biol. Chem.,263, 12461 (1988), Jpn. J. Med. Sci. Biol., 42, 77 (1989), FEBS Lett.,462, 289 (1999), J. Biol. Chem., 269, 14730-14737 (1994), J. Biol.Chem., 267, 2994 (1992), Anal. Biochem., 189, 151-162 (1990), J. Biol.Chem., 273, 433-440 (1998)]. Also, acceptor specificity of thepolypeptide of the present invention can be examined by measurementusing various acceptors.

[0241] When a cell extract is used as an enzyme sample, in order toeliminate influence of the glycosyltransferase activities possessed bythe host cell itself, a cell extract of a transformant transfected witha control vector which does not contain a DNA encoding the polypeptideof the present invention is used as a control and itsβ1,3-N-acetylglucosaminyltransferase activity is compared. Whenβ1,3-N-acetylglucosaminyltransferase activity is increased in comparisonwith the control, it can be said that the polypeptide encoded by the DNAof the present invention carried by the transformant has aβ1,3-N-acetylglucosaminyltransferase activity.

[0242] In the case of a cell or tissue expressing two or more Galβ1,3-N-acetylglucosaminyltransferases, each of the Galβ1,3-N-acetylglucosaminyltransferases cannot be specified andenzymological characteristics of each of these Galβ1,3-N-acetylglucosaminyltransferases cannot be elucidated byenzymological analysis using a cell or tissue extract. By purifying thepolypeptide of the present invention by the method described in (2),enzymological characteristics of the Galβ1,3-N-acetylglucosaminyltransferase possessed by the polypeptide can befound.

[0243] Examples of the measuring method are shown below.

[0244] (i) Method Using a Fluorescently-labeled Oligosaccharide as theAcceptor

[0245] The reaction is carried out using an oligosaccharidefluorescently-labeled by 2-aminobenzamide-labeling or pyridylaminationas an acceptor and UDP-GlcNAc as a saccharide donor, and the reactionsolution is analyzed by high performance liquid chromatography (HPLC).The 2-aminobenzamide-labeled sugar chain substrate can be prepared usingSIGMA 2AB glycan labeling kit (manufactured by Oxford Glycoscience)according to the manufacture's instructions attached to the kit. Thefluorescence-labeling by pyridylamination can be carried out in aconventional manner [Agric. Biol. Chem., 54, 2169 (1990)]. A peak whichincreases when the saccharide donor UDP-GlcNAc is added, in comparisonwith the case of no addition, is considered as a peak of a product. Theamount of the product is determined based on its fluorescence intensity,and a ratio of the product to the added acceptor is used as aβ1,3-N-acetylglucosaminyltransferase activity. The product can beidentified by coincidence of the HPLC retention time of the product withthe retention time of a standard (a labeled oligosaccharide havingstructure in which N-acetylglucosamine is added via β1,3-linkage to thereducing terminal of the acceptor oligosaccharide used in the reaction)as the index.

[0246] (ii) Method Using an Unlabeled Oligosaccharide as the Acceptor

[0247] A reaction is carried out using an unlabeled oligosaccharide asan acceptor in the same manner as in (i) and the reaction solution isanalyzed by a high speed anion exchange chromatography instead of HPLC.Using a peak with the increase amount when the saccharide donorUDP-GlcNAc is added, in comparison with the case of no addition, as apeak of a product, an amount of the product is measured, and a ratio ofthe product to the added acceptor is used as aβ1,3-N-acetylglucosaminyltransferase activity. The formed product can beidentified by coincidence of the elution time of the formed product bythe high speed anion exchange chromatography with the elution time of astandard (an oligosaccharide having structure in whichN-acetylglucosamine is added via β1,3-linkage to the reducing terminalof the acceptor oligosaccharide used in the reaction) as the index.

[0248] (iii) Method Using a Glycolipid as the Acceptor

[0249] A reaction is carried out using a glycolipid as an acceptor andUDP-[¹⁴C]GlcNAc as a saccharide donor. Glycolipids are extracted fromthe reaction solution by a reverse phase chromatography and developedusing a silica gel thin layer chromatography (TLC). The product isdetected and quantified by measuring radioactivity on the plate. A ratioof the product to the added acceptor is used as aβ1,3-N-acetylglucosaminyltransferase activity. The formed product can beidentified by coincidence of the Rf value of the product with the Rfvalue of a standard (a glycolipid having structure in whichN-acetylglucosamine is added via β1,3-linkage to the reducing terminalof the glycolipid used as the acceptor) as the index.

[0250] Also, whether or not the polypeptide of the present invention isinvolved in the in vivo sugar chain synthesis can be analyzed asfollows. Based on the description in (2), cells of a transformantprepared by introducing an expression vector containing the DNA of thepresent invention into animal cells are cultured to express thepolypeptide of the present invention. The transformant cells aresubjected to a fluorescent staining using antibodies or lectins capableof binding specifically to various sugar chains(poly-N-acetyllactosamine sugar chain, sialyl Lewis a sugar chain andsialyl Lewis c sugar chain), and then amounts of sugar chains to whichthe antibodies or lectins bind are measured using a fluorescenceactivated cell sorter (hereinafter referred to as “FACS”).

[0251] For example, as a result of confirming increase of the amount ofpoly-N-acetyllactosamine sugar chain in comparison with a transformantcell transfected with a control vector which does not contain a DNAencoding the polypeptide of the present invention, it can be found thatthe polypeptide encoded by the DNA of the present invention has aβ1,3-N-acetylglucosaminyltransferase activity involved in the synthesisof a poly-N-acetyllactosamine sugar chain.

[0252] As antibodies or lectins which recognize apoly-N-acetyllactosamine sugar chain, any antibody or lectin can beused, so long as it recognizes a poly-N-acetyllactosamine sugar chain.For example, an anti-i antibody which specifically recognizes a linearpoly-N-acetyllactosamine sugar chain (i antigen) and an anti-I antibodywhich specifically recognizes a branched poly-N-acetyllactosamine sugarchain (I antigen) [J. Biol. Chem., 254, 3221 (1979)] can be used asantibodies which specifically recognize a poly-N-acetyllactosamine sugarchain, and pokeweed mitogen (hereinafter referred to as “PWM”),Lycopersicon esculentum agglutinin (hereinafter referred to as “LEA”)and Datura stramonium agglutinin (hereinafter referred to as “DSA”) canbe used as lectins which specifically recognize apoly-N-acetyllactosamine sugar chain [J. Biol. Chem., 282, 8179-8189(1987), J. Biol. Chem., 259, 6253-6260 (1984), J. Biol. Chem., 262,1602-1607 (1987), Carbohydr. Res., 120, 187-195 (1983), Carbohydr. Res.,120, 283-292 (1983), Glycoconjugate J., 7, 323-334 (1990)].

[0253] Involvement of the polypeptide of the present invention in the invivo synthesis of sugar chains of complex carbohydrates such asglycoproteins and glycolipids can be confirmed by adding a sugar chainsynthesis inhibitor specific for the sugar chain of respective complexcarbohydrates at the time of the culturing of the transformant cells inthe above method, and then subjecting the cells in which synthesis ofthe sugar chain was inhibited to FACS analysis. For example, it can befound that the polypeptide of the present invention is involved in thesynthesis of a poly-N-acetyllactosamine sugar chain in O-linked sugarchains of glycoprotein, if the amount of poly-N-acetyllactosamine sugarchain is reduced when transformant cells are fluorescently-stained usingan antibody which recognizes a poly-N-acetyllactosamine sugar chain andthen analyzed by FACS, after culturing of the transformant cells in thepresence of an inhibitor specific for the synthesis of O-linked sugarchains of glycoprotein.

[0254] Examples of the sugar chain synthesis inhibitor specific for thesugar chains of complex carbohydrates include Benzyl-α-GalNAc which is asynthesis inhibitor of glycoprotein O-linked sugar chains, a mannosidaseII inhibitor swainsonine which acts as a synthesis inhibitor ofglycoprotein N-linked sugar chains, and a glucosylceramide synthaseinhibitor D-PDMP(D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) which acts asa synthesis inhibitor of glycolipid sugar chains.

[0255] Also, involvement of the polypeptide of the present invention inthe synthesis of glycolipid sugar chains can be confirmed by extractingglycolipids from a transformant cell prepared by introducing anexpression vector for the polypeptide of the present invention into ananimal cell and from a transformant cell prepared by introducing acontrol vector which does not contain a DNA encoding the polypeptide ofthe present invention, and analyzing and comparing compositions of bothglycolipids using a TLC plate. Extraction of glycolipids and analysis ofcompositions can be carried out according to a known method [Shujunsha,Cell Technology (Saibo Kogaku), Supplement, “Glycobiology ExperimentProtocol (Glycobiology Jikken Protocol)”; Anal. Biochem., 223, 232(1994)]. The glycolipid developed on a TLC plate is detected andquantified by orcinol staining or immunostaining which involves use ofan antibody capable of binding to specific sugar chain structure andidentified by comparing with the Rf value of a standard glycolipid. Forexample, it can be found that the polypeptide of the present inventionis involved in the synthesis of paragloboside(Galβ1-4GlcNAcβ1-3Galpl-4Glc-ceramide) and neolactohexaosylceramide(Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide), if the amountsof paragloboside and neolactohexaosylceramide are increased in atransformant cell in which the polypeptide of the present invention isexpressed, in comparison with a control transformant cell, whenparagloboside and neolactohexaosylceramide are detected byimmunostaining involving use of an antibody which recognizes theN-acetyllactosamine structure.

[0256] The polypeptide of the present invention can be used asmedicaments for treating, preventing and/or diagnosing diseases whichaccompany changes in the expression of the polypeptide of the presentinvention such as inflammatory disease, cancer and tumor metastasis.

[0257] Although the medicament comprising the polypeptide of the presentinvention can be used directly as a therapeutic agent, generally, it ispreferred to use it as a pharmaceutical preparation produced by awell-known method in the technical field of pharmaceutics by mixing itwith at least one pharmaceutically acceptable carrier. As theadministration method of the therapeutic agent, it is preferred to usethe most effective method in carrying out the treatment, and methods byoral administration or by parenteral administration such as buccal,airway, rectal, subcutaneous, intramuscular or intravenous can be used.A dosage form of the therapeutic drug include ointments, sprays,capsules, tablets, granules, syrups, emulsions, suppositoriesinjections, tapes and the like.

[0258] The preparations suitable for oral administration includeemulsions, syrups, capsules, tablets, powders, granules and the like.For example, liquid preparations such as emulsions and syrups can beproduced using, as additives, water; saccharides such as sucrose,sorbitol and fructose; glycols such as polyethylene glycol and propyleneglycol; oils such as sesame oil, olive oil and soybean oil; antisepticssuch as p-hydroxybenzoates; flavors such as strawberry flavor andpeppermint; and the like. Capsules, tablets, powders, granules and thelike can be produced using, as additives, fillers such as lactose,glucose, sucrose and mannitol; disintegrants such as starch and sodiumalginate; lubricants such as magnesium stearate and talc; binders suchas polyvinyl alcohol, hydroxypropylcellulose and gelatin; surfactantssuch as fatty acid ester; plasticizers such as glycerin; and the like.

[0259] The preparations suitable for parenteral administration includeinjections, suppositories, sprays and the like. For example, injectionscan be prepared using, for example, a carrier comprising a saltsolution, a glucose solution, or a mixture of both or the like.Suppositories can be produced using, for example, a carrier such ascacao butter, hydrogenated fat or carboxylic acid. Also, sprays can beprepared from the protein itself or using a carrier or the like whichdoes not stimulate oral and airway mucous membranes of patients and canfacilitate absorption of the protein by dispersing it as minuteparticles. The carrier includes lactose, glycerol and the like.Depending on the properties of the protein and the carrier, it ispossible to prepare pharmaceutical preparations such as aerosols and drypowders. The components exemplified as additives of oral preparationscan also be added to these parenteral preparations.

[0260] The dose or frequency of administration varies depending on theintended therapeutic effect, administration method, treating period,age, body weight and the like, but is generally from 1 μg/kg to 100mg/kg per day and per adult.

[0261] Furthermore, now that a polypeptide of the present inventionhaving a lactosylceramide β1,3-N-acetylglucosaminyltransferase activityand a DNA encoding the polypeptide have been obtained, functionalanalyses and applications as shown below become possible.

[0262] (i) Functional analysis of the polypeptide of the presentinvention by molecular biological techniques or using a knockout mouse,a transgenic mouse or the like.

[0263] (ii) Expression distribution analysis of the polypeptide of thepresent invention and DNA encoding the polypeptide.

[0264] (iii) Analysis of relationship of expression of the polypeptideof the present invention and DNA encoding the polypeptide with variousdiseases.

[0265] (iv) Diagnosis of diseases using expression of the polypeptide ofthe present invention and DNA encoding the polypeptide as the marker.

[0266] (v) Identification of type and differentiation stage of cellsusing expression of the polypeptide of the present invention and DNAencoding the polypeptide as a marker.

[0267] (vi) Screening of a compound which increases or inhibitsexpression of the polypeptide of the present invention and DNA encodingthe polypeptide or enzyme activity possessed by the polypeptide of thepresent invention.

[0268] (vii) Synthesis of useful sugar chains using the polypeptide ofthe present invention.

[0269] Examples of the use of the polypeptide of the present inventionand DNA encoding the polypeptide are specifically described as follows.

(4) Production and Application of a Sugar Chain Having Structure inwhich N-acetylglucosamine is Added to Galactose Residue via β1,3-linkageand complex carbohydrate Containing the Sugar Chain

[0270] The polypeptide of the present invention has an activity totransfer N-acetylglucosamine via β1,3-linkage to galactose residuepresent in the non-reducing terminal of an acceptor sugar chain, namelyβ1,3-N-acetylglucosaminyltransferase activity. Accordingly, thepolypeptide of the present invention can be used as a sugar chainsynthesizing agent. Examples of acceptors include i) galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose(Galβ1-4Glc), ii) an oligosaccharide having galactose,N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure in thenon-reducing terminal, and iii) a complex carbohydrate having galactose,N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure in thenon-reducing terminal. The polypeptide of the present invention has anenzyme activity to transfer N-acetylglucosamine via β1,3-linkage togalactose residue present in the non-reducing terminal of an acceptorsugar chain selected from the above i) to iii). The complex carbohydrateincludes a complex carbohydrate selected from a glycoprotein, aglycolipid, a proteoglycan, a glycopeptide, a lipopolysaccharide, apeptidoglycan, a glycoside in which a sugar chain is linked to a steroidcompound and the like. Examples of the complex carbohydrate havinggalactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure inthe non-reducing terminal include lactosylceramide, paragloboside andthe like.

[0271] Examples of the structure in which N-acetylglucosamine is addedto a galactose residue via β1,3-linkage include GlcNAcβ1-3Gal structure,GlcNAcβ1-3Galβ1-4GlcNAc structure, GlcNAcβ1-3Galβ1-3GlcNAc structure,GlcNAcβ1-3Galβ1-4Glc structure, (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAcstructure (n is an integer of 1 or more),(Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structure (n is an integer of 1 ormore) and the like.

[0272] The details are shown as follows.

[0273] (i) Process for Producing a Sugar Chain Using TransformantTransfected with DNA Encoding the Polypeptide of the Present Invention

[0274] A sugar chain having structure in which N-acetylglucosamine isadded to a galactose residue via β1,3-linkage, specifically a sugarchain comprising a saccharide selected from the group consisting ofGlcNAcβ1-3Galβ1-4Glc-ceramide, a lacto-series glycolipid (a glycolipidhaving Galβ1-3GlcNAcβ1-3Galβ1-4Glc-ceramide as a backbone), aneolacto-series glycolipid (a glycolipid havingGalβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide as a backbone), a saccharide havinga GlcNAcβ1-3Gal structure, a saccharide having GlcNAcβ1-3Galβ1-4GlcNAcstructure, a saccharide having GlcNAcβ1-3Galβ1-3GlcNAc structure, asaccharide having GlcNAcβ1-3Galβ1-4Glc structure, a saccharide having(Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc structure wherein n is 1 or moreand a saccharide having (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structurewherein n is 1 or more, or a complex carbohydrate containing the abovesugar chain, can be produced by culturing a transformant selected fromtransformants obtained in the above (3) from microorganisms, animalcells, plant cells and insect cells, in a culture medium to produce andaccumulate the sugar chain or complex carbohydrate, and then recoveringthe sugar chain or complex carbohydrate from the culture mixture.

[0275] The culturing can be carried out according to the above (3). Asugar chain having structure in which N-acetylglucosamine is added to agalactose residue via β1,3-linkage can be added to the recombinantglycoprotein by simultaneously producing the polypeptide of the presentinvention and a recombinant glycoprotein of interest (e.g., arecombinant glycoprotein for medicament) in a transformant capable ofsynthesizing sugar chains among the above transformants.

[0276] (ii) Process for Producing a Sugar Chain Using an Animal or Plantinto which DNA Encoding the Polypeptide of the Present Invention isIntroduced

[0277] Using an animal or plant obtained in the above (3), a sugar chainhaving structure in which N-acetylglucosamine is added to a galactoseresidue via β1,3-linkage or a complex carbohydrate to which the sugarchain is added can be produced according to the process of above (3).

[0278] That is, a sugar chain having structure in whichN-acetylglucosamine is added to a galactose residue via β1,3-linkage ora complex carbohydrate to which the sugar chain is added, specificallyGlcNAcβ1-3Galβ1-4Glc-ceramide, a lacto-series glycolipid (a glycolipidhaving Galβ1-3GlcNAcβ1-3Galβ1-4Glc-ceramide as a backbone), aneolacto-series glycolipid (a glycolipid havingGalβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide as a backbone), a sugar chaincomprising a saccharide selected from the group consisting of asaccharide having GlcNAcβ1-3Gal structure, a saccharide havingGlcNAcβ1-3Galβ1-4GlcNAc structure, a saccharide havingGlcNAcβ1-3Galβ1-3GlcNAc structure, a saccharide havingGlcNAcβ1-3Galβ1-4Glc structure, a saccharide having(Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc structure wherein n is 1 or moreand a saccharide having (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structurewherein n is 1 or more, or a complex carbohydrate containing the abovesugar chain, can be produced by breeding a non-human transgenic animalcarrying the DNA of the present invention to produce and accumulate thesugar chain or complex carbohydrate containing the sugar chain, and thenrecovering the product from the animal.

[0279] The sugar chain or complex carbohydrate can be produced andaccumulated, for example, in milk, egg and the like of the animal.

[0280] In the case of a plant, a sugar chain having structure in whichN-acetylglucosamine is added to a galactose residue via β1,3-linkage ora complex carbohydrate to which the sugar chain is added can beproduced, for example, by cultivating a transgenic plant comprising theDNA of the present invention to produce and accumulate the sugar chainhaving structure in which N-acetylglucosamine is added to a galactoseresidue via β1,3-linkage or complex carbohydrate to which the sugarchain is added, and then recovering the product from the plant.

[0281] (iii) Process for Producing a Sugar Chain Using the Polypeptideof the Present Invention

[0282] Using the polypeptide of the present invention obtained by theprocess described in the above (3) as an enzyme source, and galactose,an oligosaccharide having a galactose residue in the non-reducingterminal or a complex carbohydrate having a galactose residue in thenon-reducing terminal of its sugar chain as an acceptor, a reactionproduct in which N-acetylglucosamine is added via β1,3-linkage togalactose or a galactose residue present in the non-reducing terminal ofthe sugar chain can be produced in an aqueous medium by the followingprocess.

[0283] That is, a reaction product in which N-acetylglucosamine is addedvia β1,3-linkage to galactose or a galactose residue of an acceptor canbe produced by using at least one species selected from the groupconsisting of i) lactosylceramide (Galβ1-4Glc-ceramide) or paragloboside(Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide), ii) galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNac, Galβ1-3GalNAc orlactose (Galβ1-4Glc), iii) an oligosaccharide having galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose(Galβ1-4Glc) structure in the non-reducing terminal, and iv) a complexcarbohydrate having galactose, N-acetyllactosamine, Galβ1-3GlcNAc orlactose structure in the non-reducing terminal, as an acceptor, and thepolypeptide of the present invention obtained by the process describedin the above (3) as an enzyme source, allowing the acceptor, the enzymesource and N-acetylglucosamine uridine 5′-diphosphate (to be referred toas UDP-GlcNAc hereinafter) to be present in an aqueous medium to produceand accumulate the reaction product, and then recovering the reactionproduct from the aqueous medium.

[0284] The enzyme source is used at a concentration of 0.1 mU/l to10,000 U/1, preferably 1 mU/l to 1,000 U/l, by defining the activitycapable of forming 1 μmol of GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc in 1minute at 37° C. as 1 unit (U) when lacto-N-neotetraose(Galβ1-4GlcNAcβ1-3Galβ1-4Glc) is used as the substrate.

[0285] Examples of the aqueous media include water, buffer solutionssuch as phosphate buffer, carbonate buffer, acetate buffer, boratebuffer, citrate buffer and tris buffer, alcohols such as methanol andethanol, esters such as ethyl acetate, ketones such as acetone, amidessuch as acetamide, and the like. A culture medium of a microorganismused as an enzyme source can also be used as the aqueous medium. Inaddition, the culture medium of a transformant obtained by the culturingdescribed in the above (2) or the milk obtained from a non-humantransgenic animal described in the above (2) can also be used as theaqueous medium. If necessary, a surfactant or an organic solvent may beadded to the aqueous medium.

[0286] The surfactant may be any agent that can accelerate production ofa sugar chain having structure in which N-acetylglucosamine is added toa galactose residue via β1,3-linkage or a complex carbohydrate to whichthe sugar chain is added. Examples include nonionic surfactants such aspolyoxyethylene octadecylamine (e.g., Nymeen S-215, manufactured byNIPPON OIL & FATS), cationic surfactants such as cetyltrimethylammoniumbromide and alkyldimethyl benzylammonium chloride (e.g., Cation F2-40E,manufactured by NIPPON OIL & FATS), anionic surfactants such as laurylsarcosinate, tertiary amines such as alkyl dimethylamine (e.g., TertiaryAmine FB, manufactured by NIPPON OIL & FATS), and the like, which may beused alone or as a mixture of two or more thereof. The surfactant isgenerally used at a concentration of 0.1 to 50 g/l.

[0287] The organic solvent includes xylene, toluene, an aliphaticalcohol, acetone, ethyl acetate and the like, and it is generally usedat a concentration of 0.1 to 50 ml/l. As the UDP-GlcNAc, a reactionsolution produced by using activity of a microorganism or the like or aproduct purified from the reaction solution can be used, in addition tocommercially available products. The UDP-GlcNAc is used at aconcentration of 0.1 to 500 mmol/l.

[0288] Examples of the oligosaccharides having galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose(Galβ1-4Glc) structure in the non-reducing terminal, other than thosedescribed above, include Galβ1-3GalNAc, Galβ1-4GlcNAcβ1-3Galβ1-4Glc,Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc, Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc,Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4GlcNac,Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc,Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc, Galβ1-3GlcNAcβ1-3Galβ1-4Glc,Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc, Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc,Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc,Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4Glc,Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc,Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc,Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc,Galβ1-3GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4Glc,Galβ1-3GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc,Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc andGalβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc, or an oligosaccharidehaving structure of any one of these oligosaccharide structures in thenon-reducing terminal of the sugar chain. The complex carbohydrateshaving a galactose residue in the non-reducing terminal of its sugarchain include a complex carbohydrate containing a sugar chain havingstructure of any one the above oligosaccharide structure in thenon-reducing terminal of the sugar chain, a complex carbohydratecontaining an asialo complex N-linked sugar chain and the like. Specificexamples include glycolipids such as lactosylceramide(Galβ1-4Glc-ceramide) and paragloboside(Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide).

[0289] The acceptor can be used at a concentration of 0.01 to 500mmol/l.

[0290] In the production reaction, inorganic salts such as MnCl₂,β-mercaptoethanol, polyethylene glycol and the like can be added as theoccasion demands. The production reaction is carried out in an aqueousmedium at pH 5 to 10, preferably pH 6 to 8, and at 20 to 50° C. for 1 to96 hours.

[0291] A part of a sugar chain can be cut out from the sugar chain orcomplex carbohydrate produced by the above process by known enzymatictechniques or chemical techniques [Second Biochemical ExperimentationSeries (Zoku Seikagaku Jikken Koza), Vol. 4, “Method for StudyingComplex Carbohydrates (Fukugo Toshitsu Kenkyu-ho)” I, II, edited byJapanese Biochemical Society, Tokyo Kagaku Dojin (1986), GlycobiologyExperimentation Protocol (Glycobiology Jikken Protocol), edited byNaoyuki Taniguchi, Akemi Suzuki, Kiyoshi Furukawa and Kazuyuki Sugawara,Shujun-sha, (1996)].

[0292] A galactose-added sugar chain or complex carbohydrate can beproduced using the N-acetylglucosamine-added sugar chain or complexcarbohydrate obtained by the above process as an acceptor and byallowing a) the acceptor, b) GlcNAc β1,4-galactosyltransferase and c)uridine 5′-diphosphate galactose (hereinafter referred to as “UDP-Gal”)to be present in an aqueous medium to produce and accumulate a reactionproduct in which the galactose is added via β1,4-linkage to theN-acetylglucosamine residue at the non-reducing terminal of the acceptorin the aqueous medium, and recovering the galactose-added sugar chain orcomplex carbohydrate from the aqueous medium.

[0293] In addition, it is known that a poly-N-acetyllactosamine sugarchain [a sugar chain constructed by two or more repetition of(Galβ1-4GlcNAcβ1-3) structure] is synthesized by repetitive action of aGlcNAc β1,4-galactosyltransferase and a Galβ1,3-N-acetylglucosaminyltransferase. Thus, the poly-N-acetyllactosaminesugar chain can be synthesized in vitro using GlcNAcβ1,4-galactosyltransferase and the polypeptide of the present inventionhaving a Gal β1,3-N-acetylglucosaminyltransferase activity.

[0294] That is, a poly-N-acetyllactosamine sugar chain-added sugar chainor complex carbohydrate can be produced using the polypeptide of thepresent invention as an enzyme source, by allowing (a) GlcNAcβ1,4-galactosyltransferase, (b) an acceptor selected from i)lactosylceramide (Galβ1-4Glc-ceramide) or paragloboside(Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide), ii) galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose(Galβ1-4Glc), iii) an oligosaccharide having galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose(Galβ1-4Glc) structure in the non-reducing terminal, iv) a complexcarbohydrate having galactose, N-acetyllactosamine, Galβ1-3GlcNAc orlactose structure in the non-reducing terminal and v) a sugar chain orcomplex carbohydrate obtained by the process described above, (c)uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc), and (d) uridine5′-diphosphate galactose (UDP-Gal) to be present in an aqueous medium toproduce and accumulate a reaction product in which apoly-N-acetyllactosamine sugar chain is added to the non-reducingterminal of the acceptor in the aqueous medium, and recovering thepoly-N-acetyllactosamine sugar chain-added sugar chain or complexcarbohydrate from the aqueous medium.

[0295] Furthermore, a poly-N-acetyllactosamine sugar chain or a complexcarbohydrate to which the sugar chain is added can be produced byco-expressing a GlcNAc β1,4-galactosyltransferase and a DNA encoding thepolypeptide of the present invention having a Galβ1,3-N-acetylglucosaminyltransferase activity in a cell. Since GlcNAcβ1,4-galactosyltransferase is expressed in almost all cells, apoly-N-acetyllactosamine sugar chain or a complex carbohydrate to whichthe sugar chain is added can also be produced by expressing the DNAencoding the polypeptide of the present invention having a Galβ1,3-N-acetylglucosaminyltransferase activity alone in a cell.

[0296] (iv) Applications of Various Sugar Chains or ComplexCarbohydrates

[0297] For example, the following applications can be considered asapplications of the thus produced various sugar chains or complexcarbohydrates.

[0298] It is known that lacto-N-neotetraose(Galβ1-4GlcNAcβ1-3Galβ1-4Glc) and lacto-N-tetraose(Galβ1-3GlcNAcβ1-3Galβ1-4Glc) or various oligosaccharides having them asa backbone are present in human milk [Acta Paediatrica, 82, 903 (1993)].The oligosaccharides have GlcNAcβ1-3Gal structure in common. Also,oligosaccharides having a poly-N-acetyllactosamine sugar chain areincluded in the oligosaccharides. It is considered that theseoligosaccharides function to prevent babies from infection with virusesand microorganisms or to neutralize toxins. In addition, their activityto accelerate growth of Lactobacillus bifidus which is a beneficialenteric bacterium is also known. On the other hand, types ofoligosaccharides existing in the milk of animals such as cows and miceare few and are mostly lactose, and the above oligosaccharides existingin human milk are hardly present therein.

[0299] It may be industrially markedly useful if the aboveoligosaccharides contained in human milk or a milk containing them canbe produced efficiently. Since there is a possibility that thepolypeptide of the present invention having a lactosylceramideβ1,3-N-acetylglucosaminyltransferase activity is involved in thesynthesis of the above oligosaccharides contained in human milk atmammary gland, there is a possibility that it can be used in theproduction of oligosaccharides effective in treating infectious diseasesand accelerating growth of the beneficial Lactobacillus bifidus.

[0300] Since a poly-N-acetyllactosamine sugar chain contributes to thestabilization of proteins, any protein can be stabilized by artificiallyadding a poly-N-acetyllactosamine sugar chain to the protein. Also,since the clearance rate of a blood protein from the kidney becomes slowas the effective molecular weight of the protein increases, stability ofany protein in blood can be increased through its reduced clearance ratefrom the kidney by increasing its effective molecular weight byartificially adding a poly-N-acetyllactosamine sugar chain to theprotein. Also, targeting of any protein of interest into a specifiedcell can be carried out by adding a poly-N-acetyllactosamine sugarchain.

(5) Application of the DNA or Oligonucleotide of the Present Invention

[0301] Applications using the DNA or oligonucleotide of the presentinvention or derivatives thereof are described below in detail.

[0302] (i) Determination of the Expression Level of a DNA Encoding thePolypeptide of the Present Invention

[0303] Determination of the expression level of a gene encoding thepolypeptide of the present invention or detection of structural changeof the gene can be carried out by using the DNA of the present inventionor the above oligonucleotide prepared from the DNA.

[0304] The method for determining expression level of mRNA encoding thepolypeptide of the present invention or detecting structural changes ofDNA and mRNA encoding the polypeptide of the present invention includes(a) Northern blotting, (b) in situ hybridization, (c) quantitativePCR/real time PCR, (d) differential hybridization, (e) DNAmicro-array/DNA tip, (f) RNase protection assay and the like.

[0305] As the samples to be analyzed by the above methods, DNA, mRNA ortotal RNA obtained from cultured cells, various tissues, biologicalsamples such as sera and saliva, or the transformants described in (3)are used. Hereinafter, the mRNA and total RNA are called sample-derivedRNA. In addition, samples isolated as paraffin or cryostat sections fromtissues obtained from biological samples can also be used.

[0306] In the Northern blotting, the expression level of an mRNA and itsstructural change can be detected by detecting a band specificallybinding to the mRNA encoding the polypeptide of the present invention,by separating sample-derived RNA by gel electrophoresis, transferringthe sample onto a support such as a nylon filter, and then carrying outhybridization using a labeled probe prepared from the DNA of the presentinvention and washing. In the hybridization, incubation is performedunder such conditions that the mRNA in the sample-derived RNA forms astable hybrid with the probe. In order to prevent false positive, it ispreferred to carry out the hybridization and washing under highlystringent conditions. The conditions are determined by a large number offactors such as temperature, ionic strength, base composition, length ofthe probe and concentration of formamide. The factors are described, forexample, in Molecular Cloning, Second Edition.

[0307] A labeled probe used in the Northern blotting can be prepared,for example, by incorporating a radioisotope, biotin, a fluorescencegroup, a chemiluminescence group or the like into a DNA having anucleotide sequence complementary to the nucleotide sequence of the DNAof the present invention, a partial fragment of 100 nucleotides or moreof the DNA, or an oligonucleotide designed from the sequence of the DNA,using a known method (nick translation, random priming or kinasing).Since the amount of the bound labeled probe reflects the expressionlevel of the mRNA, the expression level of the mRNA can be determined bydetermining the amount of the bound labeled probe. Also, structuralchanges in the mRNA can be known by analyzing the labeled probe bindingsite.

[0308] The expression level of the mRNA can be detected by the in situhybridization in which hybridization and washing are carried out usingthe above labeled probe and a sample isolated as a paraffin or cryostatsection from a tissue obtained from a living body. In order to preventfalse positive in the in situ hybridization method, it is preferred tocarry out the hybridization and washing under highly stringentconditions. The conditions are determined by a large number of factorssuch as temperature, ionic strength, base composition, length of theprobe and concentration of formamide. These factors are described, forexample, in Molecular Cloning, Second Edition.

[0309] The mRNA can be detected according to a quantitative PCR, adifferential hybridization method, a DNA micro-array/DNA tip or the likeby using a sample-derived RNA or a cDNA synthesized from the RNA using areverse transcriptase. Hereinafter, the cDNA is called sample-derivedcDNA. A random primer or oligo(dT) primer can be used in the synthesisof cDNA.

[0310] In the quantitative PCR, a DNA fragment derived from mRNAencoding the polypeptide of the present invention is amplified by PCRusing the sample-derived cDNA as the template and primers (comprising apair of oligonucleotides of a DNA having a sequence identical tocontinues 5 to 120 nucleotides of the nucleotide sequence in the DNA ofthe present invention and a DNA having a sequence identical to continuos5 to 120 nucleotides of a nucleotide sequence complementary to thenucleotide sequence in the DNA of the present invention) designed basedon the nucleotide sequence in the DNA of the present invention. Sincethe amount of the amplified DNA fragment reflects the expression levelof the mRNA, it is possible to determine the amount of the mRNA by usinga DNA encoding actin, glyceraldehyde-3-phosphate dehydrogenase(hereinafter referred to as “G3PDH”) or the like as the internalcontrol. Also, changes in the structure of the mRNA can be detected byseparating the amplified DNA fragment by gel electrophoresis. In thisdetection method, it is preferred to use appropriate primers whichamplify the target sequence specifically and efficiently. Suchappropriate primers can be designed based on such conditions that, e.g.,hybridization between the primers and intramolecular hybridization of aprimer are not caused, that they specifically hybridize to the targetcDNA at the annealing temperature and are removed from the target cDNAunder denaturing conditions. It is necessary to quantify the amplifiedDNA fragment during the PCR when the amplified product is increasing byexponential function. Such PCRs can be detected by recovering theamplified DNA fragment produced by each reaction and quantitativelyanalyzing it by gel electrophoresis.

[0311] The principle of the real time PCR [Junko Stevens, ExperimentalMedicine (Jikken Igaku), Supplement, 15, 46-51 (1997)] is identical tothe above quantitative PCR, and the amount of amplified DNA fragment canbe detected in real time as released fluorescence quantity by PCR usingthe TaqMan probe and a forward primer and a reverse primer labeled withtwo fluorescence dyes, respectively.

[0312] Changes in the expression level of mRNA encoding the polypeptideof the present invention can be detected by carrying out hybridizationand washing on a filter or a basement such as a slide glass or siliconto which the DNA of the present invention is immobilized, using asample-derived cDNA. Methods based on such a principle include so-calleddifferential hybridization [Trends in Genetics, 7, 314-317 (1991)] andDNA micro array/DNA tip [Genome Research, 6, 639-645 (1996)]. In eachmethod, difference in the expression level of the mRNA between a controlsample and a target sample can be accurately detected by immobilizing aninternal control such as actin or G3PDH on a filter or a basement. Also,the expression level of the mRNA can be accurately determined bysynthesizing labeled cDNA molecules using respectively different labeleddNTP based on control sample- and target sample-derived RNAs, andsimultaneously hybridizing two labeled cDNA probes on one filter or onebasement.

[0313] In the RNase protection assay, a labeled antisense RNA issynthesized by firstly ligating a promoter sequence such as T7 promoteror SP6 promoter to the 3′-terminal of the DNA of the present invention,and then carrying out in vitro transcription using an RNA polymerase inthe presence of labeled rNTP. The labeled antisense RNA is hybridized toa sample-derived RNA to form an RNA-RNA hybrid, and then the hybrid isdigested with an RNase and the RNA fragment protected from the digestionis detected by forming a band by gel electrophoresis. The expressionlevel of mRNA encoding the polypeptide of the present invention can bedetermined by quantifying the thus obtained band.

[0314] The detection method described in the above can be used indetecting or diagnosing diseases which accompany changes in theexpression level of a gene encoding the polypeptide of the presentinvention. When such a detection or diagnosis is carried out, DNA, mRNAor total RNA obtained from a patient having inflammatory disease, canceror tumor metastasis, a patient having diseases which accompanies changesin the expression level of a DNA encoding the polypeptide of the presentinvention or a healthy person is used as the sample. The DNA, mRNA ortotal RNA can be obtained from biological samples such as varioustissues, sera and saliva of a patient or healthy person or from primaryculture cells obtained by preparing cells from the biological samplesand culturing them in an appropriate medium in test tubes. The range ofthe expression levels of the gene of the patients and healthy persons isdetermined by measuring and comparing the expression level of the geneencoding the polypeptide of the present invention in samples of two ormore patients and healthy persons by the above detection methods.Detection or diagnosis of diseases which accompany changes in theexpression of the gene can be carried out by comparing the expressionlevel of the gene in a sample of a test person with the expression levelin healthy persons.

[0315] Also, two types of Gal β1,3-N-acetylglucosaminyltransferases havealready been cloned other than the polypeptide of the present invention,and it is necessary to use a detection method based on the nucleotidesequence of a gene (e.g., Northern hybridization or PCR) in order todetect expression of a specific Galβ1,3-N-acetylglucosaminyltransferase. Using the DNA of the presentinvention, its expression can be accurately examined by discriminatingit from the already cloned two enzymes.

[0316] The determination of the expression level is specificallydescribed below.

[0317] Since it is considered that differentiation of blood cells andmutual recognition and migration of nerve cells are controlled by theexpression of lactosylceramide β1,3-N-acetylglucosaminyltransferase,there is a possibility that various diseases are induced by abnormalexpression of this enzyme or decrease or increase in the activity ofthis enzyme by mutation, therefore the various diseases can be diagnosedby determining the expression level of the DNA encoding the polypeptideof the present invention.

[0318] For example, since treatments of myelogenous leukemia andlymphocytic leukemia are different, it is considered that it isclinically very useful if there is a method for accuratelydiscriminating the two leukemia diseases. While lactosylceramideβ1,3-N-acetylglucosaminyltransferase activity is detected in myeloidcell lines, the activity is not detected in lymphocyte cell lines, sothat it is considered that the gene encoding the polypeptide of thepresent invention is expressed in myelogenous leukemia cells but thegene encoding the polypeptide of the present invention is not expressedin lymphocytic leukemia cells. Myelogenous leukemia and lymphocyticleukemia can be discriminated by determining the expression level of thegene encoding the polypeptide of the present invention by the Northernhybridization or PCR, by preparing mRNA, total RNA or cDNA from leukemiacells collected from a patient and using a DNA having a nucleotidesequence complementary to the nucleotide sequence in the DNA of thepresent invention, a partial fragment of 100 bp or more of the DNA, oran oligonucleotide designed from the nucleotide sequence in the DNA ofthe present invention.

[0319] (ii) Preparation and Identification of a Promoter Region and aTranscription Controlling Region of the DNA of the Present Invention

[0320] It is possible to prepare and identify a promoter region and atranscription controlling region of the DNA by using the DNA of thepresent invention as a probe according to a known method [New CellTechnology Experimentation Protocol (Shin Saibo Kogaku Jikken Protocol),edited by Antitumor Research Group, Institute of Medical Science, TheUniversity of Tokyo, published by Shujun-sha (1993)].

[0321] Screening of a genomic DNA library prepared using a chromosomalDNA isolated from mouse, rat, or human cells or tissues by a method suchas plaque hybridization is carried out using the DNA or oligonucleotide(particularly a 5′ side region of cDNA) of the present invention as theprobe, so that a promoter region and a transcription controlling regionof the mouse, rat, or human genomic DNA of the DNA of the presentinvention can be obtained. Also, the exon/intron structure of the DNAcan be found by comparing nucleotide sequence of the thus obtainedgenomic DNA and nucleotide sequence of cDNA. Also, a promoter region anda transcription controlling region of the DNA can also be obtained fromother non-human mammals using the same method.

[0322] Currently, sequences of a large number of human chromosomal geneswhose functions are unknown are registered in data bases. Thus, a humanchromosomal gene encoding the polypeptide of the present invention canbe identified and its structure can be found by comparing the sequenceof human cDNA encoding the polypeptide of the present invention withsequences of the human chromosomal genes registered in data bases. Whena chromosomal gene sequence which corresponds to the sequence of cDNA isregistered, a promoter region and exon and intron structure of achromosomal gene encoding the polypeptide of the present invention canbe determined by comparing the sequence of the cDNA with the sequence ofthe chromosomal gene.

[0323] The promoter regions include all promoter regions andtranscription controlling regions involved in the transcription of genesencoding the polypeptide of the present invention in mammal cells. Thetranscription controlling regions include regions containing an enhancersequence which enhances basal transcription of a gene encoding thepolypeptide of the present invention, a silencer sequence thatattenuates it and the like. Examples include a promoter region and atranscription controlling region functioning in cells selected fromleukocytes, nerve cells, tracheal cells, lung cells, colon cells,placental cells, neuroblastoma cells, glioblastoma cells, colon cancercells, lung cancer cells, pancreatic cancer cells, stomach cancer cellsand leukemia cells. The thus obtained promoter region and transcriptioncontrolling region can be applied to a screening method described belowand are also useful in analyzing the transcription controlling mechanismof the gene.

[0324] (iii) Detection of Mutation and Polymorphism of a DNA Encodingthe Polypeptide of the Present Invention

[0325] Since the novel β1,3-N-acetylglucosaminyltransferase of thepresent invention is also involved in the synthesis of apoly-N-acetyllactosamine sugar chain, it is considered that this enzymeis involved in the synthesis of a sialyl Le^(x) sugar chain in leukocyteand a cancer-related sugar chains in cancer cells (sialyl Lewis x sugarchain, sialyl Lewis a sugar chain, sialyl Lewis c sugar chain anddimeric Lewis a sugar chain). Accordingly, it is considered thatdiagnosis of inflammatory disease, cancer or tumor metastasis, orprediction of prognosis of cancer is possible by examining mutation andpolymorphism of the DNA of the present invention.

[0326] Also, it is able to use in diagnosis of other diseases such asfunctional abnormality of the gene based on polymorphism and mutation ofthe gene by examining relationship between polymorphism and mutation ofthe DNA of the present invention and diseases of organs in which the DNAis expressed.

[0327] The method for detecting mutation of the DNA of the presentinvention is described below.

[0328] The most distinct test for evaluating the presence or absence ofa disease-causing mutation in the DNA of the present invention is todirectly compare the DNA from a control group with the DNA from patientsof the disease.

[0329] Specifically, human biological samples such as tissues, sera andsaliva or primary culture cells established from the biological samplesare collected from a patient of a disease whose cause is a mutation inthe DNA encoding the polypeptide of the present invention and from ahealthy person, and DNA is extracted from the biological samples or theprimary culture cells (hereinafter, the DNA is called sample-derivedDNA). Next, the DNA encoding the polypeptide of the present invention isamplified by PCR using the sample-derived DNA as the template andprimers designed based on the nucleotide sequence in the DNA of thepresent invention. As another method, the DNA encoding the polypeptideof the present invention can be amplified by PCR using the above cDNAderived from the biological samples or the primary culture cells as thetemplate. The presence or absence of a mutation can be examined bycomparing the thus obtained the amplified DNA derived from a patientwith the amplified DNA derived from a healthy person. The comparingmethod includes a method for directly examining nucleotide sequence ofthe amplified DNA samples, a method for detecting a heterogeneous formedby hybridizing a DNA having a wild type sequence with a DNA having amutation (cf., the following description) and the like.

[0330] Furthermore, as a method for detecting the presence of a mutationin a DNA encoding the polypeptide of the present invention, which causesthe above diseases, a method for detecting a heteroduplex formed byhybridizing a DNA strand having a wild type allele with a DNA strandhaving a mutation allele can be used.

[0331] Examples of the method for detecting heteroduplex include (a)detection of heteroduplex by polyacrylamide gel electrophoresis [TrendsGenet., 7, 5 (1991)], (b) single strand conformation polymorphismanalysis [Genomics, 16, 325-332 (1993)], (c) chemical cleavage ofmismatches (CCM) [Human Molecular Genetics (1996), Tom Strachan andAndrew P. Read (BIOS Scientific Publishers Limited)], (d) enzymaticcleavage of mismatches [Nature Genetics, 9, 103-104 (1996)], (e)denaturing gradient gel electrophoresis [Mutat. Res., 288, 103-112(1993)] and the like.

[0332] (a) Detection of Heteroduplex by Polyacrylamide GelElectrophoresis

[0333] A DNA encoding the polypeptide of the present invention isamplified by PCR using a sample-derived DNA or a sample-derived cDNA asthe template and primers designed based on the nucleotide sequencerepresented by SEQ ID NO:2. The primers are designed in such a mannerthat a DNA of 200 bp or less is amplified. The thus amplified DNA (apatient-derived DNA or a mixture of a patient-derived amplified DNA witha healthy person-derived amplified DNA) is converted intosingle-stranded DNA by thermal denaturation and then double-stranded DNAis again formed by gradually reducing temperature. The double-strandedDNA is subjected to polyacrylamide gel electrophoresis. Whenheteroduplexs are formed, they can be detected as extra bands due toslower mobility than homologous double strands having no mutation. Theseparation performance is efficient when a special gel (Hydro-link, MDEor the like) is used. In the screening of fragments smaller than 200 bp,insertion, deletion and almost all of one nucleotide substitution can bedetected. It is preferred to carry out the heteroduplex analysis using asingle gel in combination with the single strand conformationpolymorphism analysis described below.

[0334] (b) Single Strand Conformation Polymorphism Analysis (SSCPAnalysis)

[0335] An amplified DNA prepared by the method described in (a) isdenatured and then subjected to electrophoresis using an undenaturedpolyacrylamide gel. The amplified DNA can be detected as a band bylabeling the primers with an isotope or fluorescence dye in the DNAamplification or by silver-staining the unlabeled amplification product.When a control sample is simultaneously subjected to the electrophoresisin order to clarify difference from the pattern of wild type, a fragmenthaving a mutation can be detected from the difference in mobility.

[0336] (c) Chemical Cleavage of Mismatches (CCM)

[0337] A DNA encoding the polypeptide of the present invention isamplified by PCR using a sample-derived DNA or a sample-derived cDNA asthe template and primers designed based on the nucleotide sequencerepresented by SEQ ID NO:2. Next, a labeled DNA prepared byincorporating a radioisotope or fluorescence dye into the DNA of thepresent invention is hybridized with the amplified DNA and treated withosmium tetroxide to cleave one of the chains of DNA where a mismatch isoccurred, and thus a mutation can be detected. The CCM is one of themethods having the highest sensitivity and can also be applied tosamples of a kilo base length.

[0338] (d) Enzymatic Cleavage of Mismatches

[0339] Mismatches can also be enzymaticcally cleaved by using acombination of enzyme involved in the intracellular repairing ofmismatches such as T4 phage resolvase or endonuclease VII with RNase A,instead of the osmium tetroxide of the above (c).

[0340] (e) Denaturing Gradient Gel Electrophoresis (DGGE)

[0341] An amplified DNA prepared by the method described in (c) issubjected to electrophoresis using a density gradient of a chemicalmodifier or a gel having temperature gradient. The amplified DNAfragment migrates in the gel to a position where it denatures intosingle strand, and stops the migration after the denaturation. Sincemobility of the amplified DNA in the gel differs in the presence andabsence of a mutation in the DNA, it is possible to detect the presenceof mutation. In order to increase the detection sensitivity, a poly(G:C)terminal may be added to respective primers.

[0342] As another method for detecting a mutation in the DNA, a proteintruncation test (PTT) [Genomics, 20, 1-4 (1994)] is exemplified.Frameshift mutation, splice site mutation and nonsense mutation whichcause deficiency of polypeptides can be specifically detected by thetest. Specifically, a DNA encoding the full-length polypeptide of thepresent invention is amplified by PCR using a sample-derived cDNA (asample-derived DNA can also be used when intron is not present in achromosomal gene) as the template and primers designed based on thenucleotide sequence represented by SEQ ID NO:2. In that case, a T7promoter sequence and a eucaryote translation initiation sequence areadded at the 5′-end of a primer corresponding to the N-terminal side ofthe polypeptide. A polypeptide can be produced by carrying out in vitrotranscription and translation using the amplified DNA. The presence orabsence of a mutation which causes deficiency of polypeptide can bedetected from the migrated position when the polypeptide is subjected toa gel electrophoresis. A mutation which causes a deficiency is notpresent when the migrated position of the polypeptide is present at aposition corresponding to the full-length polypeptide. On the otherhand, when there is a deficiency in the polypeptide, the polypeptidemigrates to a position smaller than the full-length polypeptide so thatposition of the mutation can be presumed by the position.

[0343] A disease induced by a mutation in regions other than the codingregion of the chromosomal gene encoding the polypeptide of the presentinvention can also be present. Since abnormality in the size andexpression level of mRNA is detected in patients of diseases caused bymutation in the non-coding regions, their abnormality can be examined byNorthern hybridization and PCR.

[0344] When the presence of a mutation in a non-coding region issuggested, the presence or absence of a mutation in the promoter region,transcription controlling region or intron region of the gene isinspected. These gene regions can be cloned using a DNA having thenucleotide sequence represented by SEQ ID NO:2 as the probe forhybridization. Moreover, sequence information on these gene regions canalso be obtained in some cases by comparing them with human chromosomalgene sequences registered in various data bases. As shown in Example 12,intron has not been present in the case of the chromosomal gene encodingthe polypeptide of the present invention. Mutations in the non-codingregions can be screened according to any one of the above methods.

[0345] Polymorphism analysis of the DNA of the present invention can becarried out by using the gene sequence information of the DNA.Specifically, gene polymorphism can be analyzed using Southern blotting,direct sequencing, PCR, DNA tip and the like [Clinical Inspection(Rinsho Kensa), 42, 1507-1517 (1998), Clinical Inspection (RinshoKensa), 42, 1565-1570 (1998)].

[0346] The thus found mutation and polymorphism can be identified asSNPs (single nucleotide polymorphism) having linkage to diseases, by astatistic treatment according to the method described in Handbook ofHuman Genetics Linkage, The John Hopkins University Press, Baltimore(1994). Also, a disease can be diagnosed by obtaining DNA samples from afamily having a clinical history of the above disease, according to theabove method, and detecting a mutation.

[0347] (iv) Inhibition of Transcription and Translation of DNA Encodingthe Polypeptide of the Present Invention

[0348] The DNA of the present invention can inhibit transcription ortranslation of a DNA encoding the protein of the present invention byusing antisense RNA/DNA techniques [Bioscience and Industry, 50, 322(1992), Chemistry, 46, 681 (1991), Biotechnology, 9, 358 (1992), Trendsin Biotechnology, 10, 87 (1992), Trends in Biotechnology, 10, 152(1992), Cell Technology, 16, 1463 (1997)], triple helix techniques[Trends in Biotechnology, 10, 132 (1992)] and the like. For example,production of the polypeptide of the present invention can be inhibitedby administering the DNA or oligonucleotide of the present invention.That is, each of transcription of a DNA encoding the polypeptide of thepresent invention or translation of a mRNA encoding the polypeptide ofthe present invention can be inhibited by using the DNA oroligonucleotide of the present invention or derivatives thereof. Theinhibition method can be used as medicaments for treating or preventingdiseases which accompany changes in the expression of a DNA encoding thepolypeptide of the present invention such as inflammatory disease,cancer and tumor metastasis.

[0349] It is considered that the polypeptide of the present invention isinvolved in the synthesis of neolacto-series glycolipids andlacto-series glycolipids. Thus, it is considered that cancer can betreated by inhibiting transcription of the DNA of the present inventionand translation of a mRNA encoding the polypeptide.

[0350] Also, when mechanisms of the above inflammatory reactions andmetastases are taken into consideration, it can be expected to inhibitinflammatory reactions and prevent metastases by inhibiting expressionof a poly-N-acetyllactosamine sugar chain on leukocytes and cancercells. There is a possibility that expression of apoly-N-acetyllactosamine sugar chain in leukocytes and cancer cells canbe inhibited by inhibiting transcription of the DNA of the presentinvention and translation of a mRNA encoding the polypeptide.

[0351] Furthermore, it is considered that Galβ1,3-N-acetylglucosaminyltransferases involved in the synthesis of apoly-N-acetyllactosamine sugar chain in specific leukocytes and cancercells are different. Since there is a possibility that alactosylceramide β1,3-N-acetylglucosaminyltransferase is involved in thesynthesis of a poly-N-acetyllactosamine sugar chain in specificleukocytes and cancer cells, it is considered that synthesis of apoly-N-acetyllactosamine sugar chain on specific leukocytes and cancercells expressing the polypeptide can be specifically inhibited byinhibiting transcription of the DNA of the present invention andtranslation of a mRNA encoding the polypeptide.

[0352] (v) Medicament Comprising the DNA or Oligonucleotide of thePresent Invention

[0353] A medicament comprising the DNA of the present invention, apartial fragment of the DNA or the oligonucleotide of the presentinvention can be prepared and administered according to the method for amedicament comprising the polypeptide of the present invention describedin (3).

(6) Production of Antibody which Recognizes the Polypeptide of thePresent Invention

[0354] (i) Production of Polyclonal Antibody

[0355] A polyclonal antibody can be produced by using the purifiedsample of the full-length or a partial fragment of the polypeptideobtained by the method of the above (3) or a peptide having an aminoacid of a part of the polypeptide of the present invention as theantigen and administering it to an animal.

[0356] Rabbits, goats, rats, mice, hamsters and the like can be used asthe animal to be administered. It is preferred that the dose of theantigen is 50 to 100 μg per one animal. When a peptide is used, it ispreferred to use the peptide as the antigen after conjugating it to acarrier protein such as keyhole limpet haemocyanin or bovinethyroglobulin by a covalent bond. The peptide used as the antigen can besynthesized using a peptide synthesizer.

[0357] The antigen is administered 3 to 10 times at one-to two-weekintervals after the first administration. Three to seven days after eachadministration, a blood sample is collected from the venous plexus ofthe fundus of the eye, and the serum is tested by enzyme immunoassay[Enzyme-linked Immunosorbent Assay (Koso Meneki Sokuteiho) (ELISA),published by Igaku Shoin, (1976), Antibodies—A Laboratory Manual, ColdSpring Harbor Laboratory (1988)] and the like as to whether it isreactive with the antigen used for immunization.

[0358] A polyclonal antibody can be obtained by collecting a serumsample from non-human mammal in which the serum showed a sufficientantibody titer against the antigen used for immunization, and separatingand purifying the serum.

[0359] Examples of the method for its separation and purificationinclude centrifugation, salting out with 40 to 50% saturated ammoniumsulfate, caprylic acid precipitation [Antibodies—A Laboratory Manual,Cold Spring Harbor Laboratory (1988)] and chromatography using aDEAE-Sepharose column, an anion exchange column, a protein A- orG-column, a gel filtration column or the like, which may be used aloneor in combination.

[0360] (ii) Production of Monoclonal Antibody

[0361] (a) Preparation of Antibody Producing Cells

[0362] A rat whose serum showed a sufficient antibody titer against thepartial fragment polypeptide of the polypeptide of the present inventionused in the immunization is used as the supply source of antibodyproducing cells.

[0363] Three to seven days after the final administration of the antigensubstance to the rat which showed the sufficient antibody titer, thespleen is excised. The spleen is cut to pieces in MEM (manufactured byNissui Pharmaceutical), and cells are loosened using a pair of forcepsand centrifuged at 1,200 rpm for 5 minutes and then the supernatant isdiscarded. Splenocytes in the thus obtained precipitation fraction aretreated with a Tris-ammonium chloride buffer (pH 7.65) for 1 to 2minutes for eliminating erythrocytes and then washed three times withMEM, and the thus obtained splenocytes are used as the antibodyproducing cells.

[0364] (b) Preparation of Myeloma Cells

[0365] As myeloma cells, established cell lines obtained from mouse orrat are used.

[0366] Examples include 8-azaguanine-resistant mouse (BALB/c-derived)myeloma cell lines P3-X63Ag8-U1 (hereinafter referred to as “P3-U1”)[Curr. Topics Microbiol. Immunol., 81, 1 (1978), Eur. J. Immunol., 6,511 (1976)], SP2/O-Ag14 (SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653(653) [J. Immunol., 123, 1548 (1979)], P3-X63-Ag8 (X63) [Nature, 256,495 (1975)] and the like.

[0367] The cell lines are subcultured in an 8-azaguanine medium[prepared by supplementing RPMI-1640 medium with glutamine (1.5 mmol/l),2-mercaptoethanol (5×10⁻⁵ mol/l), gentamicin (10 μg/ml) and fetal calfserum (FCS) (manufactured by CSL, 10%) and further supplementing theresulting medium (hereinafter referred to as “normal medium”) with8-azaguanine (15 μg/ml)], and they are cultured in the normal medium 3to 4 days before the cell fusion, and 2×10⁷ or more of the cells areused for the cell fusion.

[0368] (c) Preparation of Hybridoma

[0369] The antibody producing cells obtained in (a) and the myelomacells obtained in (b) are washed thoroughly with MEM or PBS (1.83 g ofdisodium hydrogenphosphate, 0.21 g of potassium dihydrogenphosphate and7.65 g of sodium chloride, 1 liter of distilled water, pH 7.2) and mixedin a proportion of antibody producing cells: myeloma cells=5 to 10:1,and the mixture is centrifuged at 1,200 rpm for 5 minutes and then thesupernatant is discarded.

[0370] Cells in the thus obtained precipitation fraction are thoroughlyloosened, a mixture of 2 g of polyethylene glycol-1000 (PEG-1000), 2 mlof MEM and 0.7 ml of dimethyl sulfoxide (DMSO) is added to the cells inan amount of 0.2 to 1 ml per 10⁸ antibody producing cells under stirringat 37° C., and then 1 to 2 ml of MEM is added several times at 1 to 2minute intervals. After the addition, the whole volume is adjusted to 50ml by adding MEM.

[0371] After the thus prepared solution is centrifuged at 900 rpm for 5minutes, the supernatant is discarded. Cells in the thus obtainedprecipitation fraction are gently loosened and then suspended in 100 mlof HAT medium [prepared by supplementing the normal medium withhypoxanthine (10⁻⁴ mol/l), thymidine (1.5×10⁻⁵ mol/l) and aminopterin(4×10⁻⁷ mol/l)] by repeated drawing up into and discharging from ameasuring pipette.

[0372] The suspension is dispensed in 100 μl/well portions into a96-well culture plate and cultured at 37° C. for 7 to 14 days in a 5%CO₂ incubator. After culturing, a portion of the culture supernatant istaken out and subjected to an enzyme immunoassay described in[Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter14 (1988)] or the like, and hybridomas which specifically react with thepartial fragment polypeptide of the polypeptide of the present inventionare selected.

[0373] As an example of the enzyme immunoassay, the following method isshown.

[0374] The partial fragment polypeptide of the polypeptide of thepresent invention used as the antigen in the immunization is coated onan appropriate plate, allowed to react with a first antibody, namely ahybridoma culture supernatant or the purified antibody obtained in thefollowing (d), further allowed to react with a second antibody, namelyan anti-rat or anti-mouse immunoglobulin antibody labeled with biotin,an enzyme, a chemiluminescence substance, a radioactive compound or thelike and then subjected to the reaction corresponding to the usedlabeled substance, and those which react specifically with thepolypeptide of the present invention are selected as hybridomas thatproduce the monoclonal antibody for the polypeptide of the presentinvention.

[0375] Cloning is repeated twice using the hybridomas according to alimiting dilution method [HT medium (a medium prepared by eliminatingaminopterin from HAT medium) for the first and the normal medium for thesecond], and those in which high antibody titer is constantly observedare selected as hybridomas that produce an anti-polypeptide antibodyagainst the polypeptide of the present invention.

[0376] (d) Preparation of Monoclonal Antibody

[0377] The hybridoma cells capable of producing a monoclonal antibodyagainst the polypeptide of the present invention obtained in (c) areinjected into the abdominal cavity of 8 to 10-week-old mice or nude micetreated with pristane [by intraperitoneal administration of 0.5 ml of2,6,10,14-tetramethylpentadecane (pristane), followed by feeding for 2weeks) at a dose of 5 to 20×10⁶ cells per animal. The hybridoma causesascites tumor in 10 to 21 days. The ascitic fluid is collected from theascites tumor-caused mice and centrifuged at 3,000 rpm for 5 minutes toremove the solid matter. The monoclonal antibody can be obtained bypurifying it from the thus obtained supernatant by the same method usedin the purification of a polyclonal antibody.

[0378] The subclass of the antibody is determined using a mousemonoclonal antibody typing kit or a rat monoclonal antibody typing kit.The amount of the protein is calculated by the Lowry method or from theabsorbance at 280 nm.

[0379] (e) Neutralizing Antibody

[0380] A neutralizing antibody having a property to inhibit activity ofthe polypeptide of the present invention by binding to the polypeptideof the present invention is included in the antibody of the presentinvention. In measuring the activity of the polypeptide of the presentinvention by the method described in (3) by adding the antibody obtainedin the above, when the activity of the polypeptide of the present.invention is reduced in comparison with a case of not adding theantibody, it can be confirmed that the antibody is a neutralizingantibody.

(7) Application of the Antibody of the Present Invention

[0381] (i) Detection and Quantification of the Polypeptide of thePresent Invention

[0382] The polypeptide of the present invention or a cell or tissuecontaining the polypeptide can be immunologically detected by anantigen-antibody reaction using an antibody which specificallyrecognizes the polypeptide of the present invention. The detectionmethod can be used in the diagnosis of diseases which accompany changesin the expression of the polypeptide of the present invention such asinflammatory disease, cancer and tumor metastasis. The detection methodcan also be used in the quantification of a protein.

[0383] Examples of the immunological detection and quantificationmethods include fluorescent antibody technique, enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA),immunohistochemistry such as immunohistostaining and immunocytostaining(ABC method, CSA method, etc.), Western blotting, dot blotting,immunoprecipitation, sandwich ELISA [Monoclonal Antibody ExperimentationManual (Tan Clone Kotai Jikken Manual), Kodansha Scientific (1987),Second Biochemical Experimentation Series (Zoku Seikagaku Jikken Koza),Vol. 5, “Method for Immuno-biochemical Research (Men-eki SeikagakuKenkyu-ho)” Tokyo Kagaku Dojin (1986)] and the like.

[0384] Fluorescent antibody technique is a method in which the antibodyof the present invention is allowed to react with a microorganism, ananimal cell or insect cell or a tissue, which expressed the polypeptideof the present invention inside or outside of the cell, and furtherallowed to react with an anti-mouse IgG antibody or a fragment thereoflabeled with a fluorescence substance such as fluorescein isothiocyanate(FITC), and then the fluorescence dye is measured using a flowcytometer.

[0385] Enzyme-linked immunosorbent assay (ELISA) is a method in whichthe antibody of the present invention is allowed to react with amicroorganism, an animal cell or insect cell or a tissue, whichexpressed the polypeptide inside or outside the cell, and furtherallowed to react with an anti-mouse IgG antibody or a binding fragmentthereof labeled with an enzyme such as peroxidase or biotin, and thenthe chromogenic dye is measured using an absorptiometer.

[0386] Radioimmunoassay (RIA) is a method in which the antibody of thepresent invention is allowed to react with a microorganism, an animalcell or insect cell or a tissue, which expressed the polypeptide insideor outside the cell, and further allowed to react with an anti-mouse IgGantibody or a fragment thereof labeled with a radioisotope, and then theisotope is measured using a scintillation counter or the like.

[0387] Immunocytostaining or immunohistostaining is a method in which anantibody capable of specifically recognizing the polypeptide is allowedto react with a microorganism, an animal cell or insect cell or atissue, which expressed the polypeptide inside or outside the cell, andfurther allowed to react with an anti-mouse IgG antibody or a fragmentthereof labeled with a fluorescence substance such as FITC or an enzymesuch as peroxidase or biotin, and then the cell is observed under amicroscope.

[0388] Western blotting is a method in which a lysate of amicroorganism, an animal cell or insect cell or a tissue, whichexpressed the polypeptide inside or outside of the cell, is separated bySDS-polyacrylamide gel electrophoresis [Antibodies—A Laboratory Manual,Cold Spring Harbor Laboratory (1988)], and the gel is blotted on a PVDFmembrane or nitrocellulose membrane, allowed to react with an antibodycapable of specifically recognizing the polypeptide on the membrane andfurther allowed to react with an anti-mouse IgG antibody or a fragmentthereof labeled with a fluorescence substance such as FITC or an enzymesuch as peroxidase or biotin, and then a reaction corresponding to thelabeled substance is carried out.

[0389] The dot blotting is a method in which a lysate of amicroorganism, an animal cell or insect cell or a tissue, whichexpressed the polypeptide inside or outside the cell, is blotted on anitrocellulose membrane, allowed to react with the antibody of thepresent invention on the membrane and further allowed to react with ananti-mouse IgG antibody or a binding fragment thereof labeled withfluorescence substance such as FITC or an enzyme such as peroxidase orbiotin, and then a reaction corresponding to the labeled substance iscarried out.

[0390] The immunoprecipitation is a method in which a lysate of amicroorganism, an animal cell or insect cell or a tissue, whichexpressed the polypeptide inside or outside the cell, is allowed toreact with an antibody capable of specifically recognizing thepolypeptide, and then an antigen-antibody complex is precipitated byadding a carrier such as protein G-Sepharose having the ability tospecifically bind to immunoglobulin.

[0391] The sandwich ELISA is a method in which a lysate of amicroorganism, an animal cell or insect cell or a tissue, whichexpressed the polypeptide of the present invention inside or outside thecell, is allowed to react with a plate to which an antibody capable ofspecifically recognizing the polypeptide is adsorbed, and furtherallowed to react with an antibody (having an antigen recognizing sitedifferent from the above antibody) labeled with a fluorescence substancesuch as FITC or an enzyme such as peroxidase or biotin, whichspecifically recognizes the polypeptide of the present invention, andthen a reaction corresponding to the labeled substance is carried out.

[0392] Also, two types of Gal β1,3-N-acetylglucosaminyltransferases havealready been cloned in addition to the polypeptide of the presentinvention, and it is necessary to carry out an immunological detectionusing a specific antibody for detecting expression of a specified Galβ1,3-N-acetylglucosaminyltransferase. Accordingly, the antibody of thepresent invention makes it possible to examine the expression of thepolypeptide of the present invention accurately.

[0393] (ii) Application to Diagnosis and Treatment of InflammatoryDiseases and Cancers

[0394] Identification of changes in the expression level of thepolypeptide in human biological samples and human derived primaryculture cells, and structural changes of the expressed polypeptide isuseful for checking the danger of causing future onset of diseases andthe cause of diseases already developed.

[0395] It is suggested that the polypeptide of the present inventioninvolved in the synthesis of neolacto-series glycolipids andlacto-series glycolipids and the synthesis of a poly-N-acetyllactosaminesugar chain in leukocytes and cancer cells. Accordingly, diagnosis,prevention and treatment of inflammatory diseases and malignancy ofcancers become possible by examining the expression level of thepolypeptide of the present invention in leukocytes and cancer cells, andby controlling activity of the polypeptide of the present inventionusing the neutralizing antibody of the present invention.

[0396] Also, since it is considered that differentiation of blood cellsand mutual recognition and migration of nerve cells are controlled bythe expression of lactosylceramide β1,3-N-acetylglucosaminyltransferase,there is a possibility that various diseases are induced by abnormalexpression of this enzyme, and decrease and increase of activity of thisenzyme by mutation. Thus, diseases relating to differentiation of bloodcells or mutual recognition and migration of nerve cells can bediagnosed, prevented and treated by examining the expression level ofthe polypeptide of the present invention in blood cells or nerve cells,or by controlling activity of the polypeptide of the present inventionusing the neutralizing antibody of the present invention.

[0397] For example, since treatments of myelogenous leukemia andlymphocytic leukemia are different, it is considered that it isclinically very useful if there is a method for accuratelydiscriminating these two leukemia diseases. While a lactosylceramideβ1,3-N-acetylglucosaminyltransferase activity is detected in myeloidcell lines, the activity is not detected in lymphocyte cell lines, sothat it is considered that the gene encoding the polypeptide of thepresent invention is expressed in myelogenous leukemia cells but thegene encoding the polypeptide of the present invention is not expressedin lymphocytic leukemia cells. It is possible to discriminatemyelogenous leukemia and lymphocytic leukemia by detecting, using theantibody of the present invention, expression of this enzyme protein inleukemia cells collected from patients.

[0398] In addition, it is considered that Galβ1,3-N-acetylglucosaminyltransferases involved in the synthesis of apoly-N-acetyllactosamine sugar chain in specific leukocytes and cancercells are different. There is a possibility that lactosylceramideβ1,3-N-acetylglucosaminyltransferase is involved in the synthesis of apoly-N-acetyllactosamine sugar chain in specific leukocytes and cancercells, therefore, by inhibiting a lactosylceramideβ1,3-N-acetylglucosaminyltransferase activity of the polypeptide of thepresent invention using a neutralizing antibody, synthesis of apoly-N-acetyllactosamine sugar chain on specific leukocytes and cancercells expressing the polypeptide can be specifically inhibited.

[0399] Examples of the diagnosing methods by detecting expression leveland structural changes of the polypeptide include the above fluorescentantibody technique, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), immunohistochemistry such as immunohistostainingand immunocytostaining (ABC method, CSA method, etc.), Western blotting,dot blotting, immunoprecipitation, sandwich ELISA and the like.

[0400] The samples diagnosed by the above methods include biologicalsamples, such as tissues, blood, sera, urine, feces and saliva,collected from patients of diseases which are known to accompany changesin the expression of the polypeptide of the present invention such asinflammatory disease, cancer or tumor metastasis, as such, or cells andcell extracts prepared from the biological samples are used.Furthermore, samples isolated as paraffin or cryostat sections fromtissues obtained from biological samples can also be used.

[0401] The method for detecting immunologically includes ELISA andfluorescent antibody technique which use a microtiter plate, Westernblot technique, immunohistostaining and the like.

[0402] Examples of the method for quantifying immunologically include asandwich ELISA involving use of two monoclonal antibodies againstdifferent epitopes among the antibodies capable of reacting with thepolypeptide of the present invention in a liquid phase and aradioimmunoassay involving use of he polypeptide of the presentinvention labeled with a radioisotope such as ¹²⁵I and an antibody whichrecognizes the polypeptide of the present invention.

[0403] Medicaments comprising the antibody of the present invention canbe prepared and administered according to the methods for medicamentscomprising the polypeptide of the present invention described in (3).

(8) Method for Preparing Recombinant Virus Vector which Produces thePolypeptide of the Present Invention

[0404] A method for preparing a recombinant virus vector for theproduction of the polypeptide of the present invention in specific humantissues is described below.

[0405] A DNA fragment having an appropriate length containing a regionmoiety encoding the polypeptide is prepared based on a full-length cDNAof the DNA of the present invention, if necessary. Examples include aDNA comprising the nucleotide sequence represented by SEQ ID NO:2, a DNAcomprising a nucleotide sequence of positions 135 to 1,268 in thenucleotide sequence represented by SEQ ID NO:2, a DNA comprising anucleotide sequence of positions 249 to 1,268 in the nucleotide sequencerepresented by SEQ ID NO:2 and the like.

[0406] A recombinant virus vector is constructed by inserting thefull-length cDNA or a fragment of the DNA into the downstream of thepromoter in a virus vector.

[0407] In the case of an RNA virus vector, a recombinant virus vector isconstructed by preparing a CRNA homologous to the full-length cDNA ofthe gene of the present invention or an RNA fragment homologous to anappropriate length DNA fragment containing a region encoding thepolypeptide, and inserting it into the downstream of the promoter in avirus vector. As the RNA fragment, a single-stranded chain of either oneof a sense chain or an antisense chain is selected in response to thetype of virus vector, in addition to a double-stranded chain. Forexample, an RNA homologous to the sense chain is selected in the case ofa retrovirus vector, while an RNA homologous to the antisense chain isselected in the case of a Sendai virus vector.

[0408] The recombinant virus vector is introduced into a packaging cellsuited for the vector.

[0409] All cells which can supply a protein encoded by a gene necessaryfor the packaging of a virus deficient in the corresponding recombinantvirus vector can be used as the packaging cell, and, e.g., humankidney-derived HEK293 cell, mouse fibroblast-derived NIH3 T3 or the likewhich expressed the following proteins can be used.

[0410] Examples of the protein supplied by the packaging cell includeproteins such as mouse retrovirus-derived gag, pol and env in the caseof a retrovirus vector; proteins such as HIV virus-derived gag, pol,env, vpr, vpu, vif, tat, rev and nef in the case of a lentivirus vector;proteins such as adenovirus-derived E1A and E1B in the case of anadenovirus vector; proteins such as Rep (p5, p19, p40) and Vp (Cap) inthe case of adeno-associated virus; and proteins such as NP, P/C, L, M,F and HN in the case of Sendai virus.

[0411] The virus vector includes those which can produce a recombinantvirus in the above packaging cell and contain a promoter at such aposition that the DNA of the present invention can be transcribed in atarget cell. The plasmid vector includes MFG [Proc. Natl. Acad. Sci.USA, 92, 6733-6737 (1995)], pBabePuro [Nucleic Acids Res., 18, 3587-3596(1990)], LL-CG, CL-CG, CS-CG and CLG [Journal of Virology, 72, 8150-8157(1998)], pAdex1 [Nucleic Acids Res., 23, 3816-3821 (1995)] and the like.

[0412] As the promoter, any promoter can be used, so long as it can workfor expression in human tissues. Examples include IE (immediate early)gene promoter of cytomegalovirus (human CMV), SV40 early promoter,retrovirus promoter, metallothionein promote, heat shock proteinpromoter, SRα promoter and the like. Furthermore, an enhancer of the IEgene of human CMV may be used together with the promoter.

[0413] The method for introducing a recombinant virus vector into apackaging cell includes a calcium phosphate method (Japanese PublishedUnexamined Patent Application No. 227075/90), a lipofection method[Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like.

[0414] The virus vector which comprises the DNA of the present inventionor an RNA comprising a sequence homologous to the DNA can be used as amedicament for treating or preventing diseases which accompany changesin the expression of the polypeptide of the present invention or a DNAencoding the polypeptide such as inflammatory disease, cancer ormetastases, as a gene therapy agent described later.

(9) Application to Screening Methods

[0415] The polypeptide of the present invention has aβ1,3-N-acetylglucosaminyltransferase activity, namely, an activity totransfer N-acetylglucosamine via β1,3-linkage to a galactose residuepresent in the non-reducing terminal of a sugar chain. Specifically, ithas an activity to transfer N-acetylglucosamine via β1,3-linkage to agalactose residue present in the non-reducing terminal of a sugar chainof an acceptor selected from i) galactose, N-acetyllactosamine(Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose (Galβ1-4Glc), ii) anoligosaccharide having galactose, N-acetyllactosamine, Galβ1-3GlcNAc orlactose structure in the non-reducing terminal, and iii) a complexcarbohydrate having galactose, N-acetyllactosamine, Galβ1-3GlcNAc orlactose structure in the non-reducing terminal, includinglactosylceramide and paragloboside. Thus, a compound which changes thisactivity can be screened by contacting the polypeptide of the presentinvention with a sample to be tested.

[0416] Also, since the polypeptide of the present invention is involvedin the synthesis of neolacto-series glycolipids and lacto-seriesglycolipids or the synthesis of a poly-N-acetyllactosamine sugar chainin various cells, it is possible to increase or decrease the synthesizedamount of a neolacto-series glycolipid, a lacto-series glycolipid orpoly-N-acetyllactosamine sugar chain in cells by use of a compoundcapable of enhancing or inhibiting theβ1,3-N-acetylglucosaminyltransferase activity of the polypeptide.

[0417] Furthermore, a compound which accelerates or inhibits thetranscription step of a gene encoding the polypeptide or the translationstep of the transcripts into protein can control a synthesized amount ofa neolacto-series glycolipid, a lacto-series glycolipid orpoly-N-acetyllactosamine sugar chain in cells by controlling expressionof the polypeptide.

[0418] Since it is known that a sialyl Lewis x sugar chain and a sialylLewis a sugar chain existing on the poly-N-acetyllactosamine sugar chainare ligands of selecting, it is considered that a compound capable ofdecreasing the synthesized amount of a poly-N-acetyllactosamine sugarchain is useful for anti-inflammation and tumor metastasis inhibition.On the other hand, a compound which increases the synthesized amount ofa poly-N-acetyllactosamine sugar chain is considered to be useful in thesynthesis of a poly-N-acetyllactosamine sugar chain and production of acomplex carbohydrate to which a poly-N-acetyllactosamine sugar chain isadded.

[0419] The compounds can be obtained by the following methods (i) to(vi).

[0420] (i) A compound having an activity to increase or decrease aβ1,3-N-acetylglucosaminyltransferase activity is selected and obtainedby measuring the β1,3-N-acetylglucosaminyltransferase activity using themethod described in (3), in the presence of a compound to be tested andusing the polypeptide of the present invention prepared using the methoddescribed in the above (3) (a purified product or a cell extract orculture supernatant of a transformant expressing the polypeptide) as anenzyme.

[0421] (ii) A compound having an activity to increase or decrease theamount of a poly-N-acetyllactosamine sugar chain is selected andobtained by culturing a cell capable of expressing the polypeptide ofthe present invention or the transformant described in the above (2)using the culturing method described in the above (2) for 2 hours to 1week in the presence of a compound to be tested, and then measuring theamount of paragloboside or a poly-N-acetyllactosamine sugar chain on thecell surface by use of an antibody capable of recognizing paraglobosideor an antibody capable of recognizing poly-N-acetyllactosamine sugarchain (anti-i antibody or anti-I antibody) or a lectin (LEA, PWM orDEA).

[0422] Examples of the measuring methods involving use of the aboveantibody or lectin include detection methods involving use ofmicrotiter-aided ELISA, fluorescent antibody technique, Western blottechnique, immunohistostaining and the like. The measurement can be alsocarried out by use of FACS.

[0423] (iii) A compound having an activity to increase or decrease aβ1,3-N-acetylglucosaminyltransferase activity or an activity to increaseor decrease the amount of paragloboside or a poly-N-acetyllactosaminesugar chain is selected and obtained by synthesizing a large number ofpeptides constituting parts of the polypeptide in a high density onplastic pins or a certain solid support, efficiently screening compoundswhich selectively bind to the peptides (WO 84/03564), and then carryingout by the above method (i) or (ii).

[0424] (iv) A compound having an activity to increase or decrease theamount of the polypeptide of the present invention is selected andobtained by culturing a cell capable of expressing the polypeptide bythe culturing method described in the above (2) for 2 hours to 1 week inthe presence of a compound to be tested, and then measuring the amountof the polypeptide in the cell by use of the antibody of the presentinvention described in the above (6).

[0425] Increased or decreased expression of the polypeptide can bedetected by the fluorescent antibody technique, enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA),immunohistochemistry such as immunohistostaining method andimmunocytostaining (ABC method, CSA method, etc.), Western blotting, dotblotting, immunoprecipitation or sandwich ELISA described in the above(7).

[0426] (v) A compound having an activity to increase or decrease theamount of transcripts of a DNA encoding the polypeptide of the presentinvention is selected and obtained by culturing a cell capable ofexpressing the polypeptide by the culturing method described in theabove (2) for 2 hours to 1 week in the presence of a compound to betested, and then measuring the amount of the transcripts in the cell bythe methods described in the above (4) such as Northern hybridization,PCR or RNase protection assay.

[0427] (vi) A plasmid which carries a DNA obtained by ligating areporter gene to the downstream of a promoter described in the above (2)is prepared by a known method and introduced into an animal celldescribed in the above (2) according to the method described in theabove (2), thereby obtaining a transformant. Thereafter, by culturingthe transformant by the culturing method described in the above (2) for2 hours to 1 week in the presence of a compound to be tested, and thenmeasuring expression level of the reporter gene in the cell using knownmethods [New Cell Technology Experimentation Protocol (Shin Saibo KogakuJikken Protocol), edited by Antitumor Research Group, Institute ofMedical Science, The University of Tokyo, published by Shujun-sha(1993), Biotechniques, 20, 914 (1996), J. Antibiotics, 49, 453 (1996),Trends in Biochemical Sciences, 20, 448 (1995), Cell Technology, 16, 581(1997)], a compound having an activity to increase or decrease theexpression level is selected and obtained.

[0428] Examples of the reporter genes include a chloramphenicolacetyltransferase (CAT) gene, a β-galactosidase gene, a β-lactamasegene, a luciferase gene, a green fluorescent protein gene and the like.

[0429] It is considered that the polypeptide of the present invention isinvolved in the synthesis of neolacto-series glycolipids andlacto-series glycolipids in cancer cells. Thus, it is considered thatcancer can be treated by inhibiting synthesis of neolacto-seriesglycolipids and lacto-series glycolipids in cancer cells by use of acompound which inhibits expression of the polypeptide or DNA of thepresent invention and is obtained by the above screening.

[0430] Also, when mechanisms of the above inflammatory reactions andmetastases are taken into consideration, it can be expected to inhibitan inflammatory reaction and prevent tumor metastasis by inhibitingexpression of a poly-N-acetyllactosamine sugar chain on leukocytes andcancer cells. It is considered that inhibition of inflammatory reactionsand prevention of metastases become possible by inhibiting expression ofa poly-N-acetyllactosamine sugar chain on leukocytes and cancer cellsusing a compound which inhibits expression of the polypeptide or DNA ofthe present invention and is obtained by the above screening.

[0431] It is considered that Gal β1,3-N-acetylglucosaminyltransferasesinvolved in the synthesis of a poly-N-acetyllactosamine sugar chain inspecific leukocytes and cancer cells are different. Since there is apossibility that lactosylceramide β1,3-N-acetylglucosaminyltransferaseis involved in the synthesis of a poly-N-acetyllactosamine sugar chainin specific leukocytes and cancer cells, it can be expected that by acompound which inhibits expression of the DNA or polypeptide of thepresent invention, the synthesis of a poly-N-acetyllactosamine sugarchain in specific leukocytes and cancer cells expressing the polypeptidecan be specifically inhibited.

(10) Preparation of Non-human Knockout Animal

[0432] Using a vector containing the DNA of the present invention, amutant clone in which a DNA encoding the polypeptide of the presentinvention on the chromosome in embryonic stem cells of an objectiveanimal such as cow, sheep, goat, pig, horse, domestic fowl or mouse isinactivated or substituted by any sequence by known homologousrecombination techniques [e.g., Nature, 326, 6110, 295 (1987), Cell, 51,3, 503 (1987)] can be prepared [e.g., Nature, 350, 6315, 243 (1991)].

[0433] Using the embryonic stem cell clone prepared in this manner, achimeric individual comprising the embryonic stem cell clone and anormal cell can be prepared by a method such as an injection chimeramethod into blastocyst of fertilized egg of an animal or an assemblychimera method. An individual having an optional mutation in the DNAencoding the polypeptide of the present invention on chromosomes of thewhole body cells can be obtained by crossing this chimeric individualwith a normal individual, and a homologous individual (non-humanknockout animal) in which a mutation is introduced into both ofhomologous chromosomes can be obtained by further crossing theindividuals.

[0434] In this way, a mutation can be introduced into any position ofthe DNA encoding the polypeptide of the present invention on thechromosome of an animal individual. For example, mutation such asnucleotide substitution, deletion or insertion can be introduced into atranslation region of the DNA encoding the polypeptide of the presentinvention on the chromosome.

[0435] Also, the degree, time, tissue specificity and the like of theexpression can be modified by introducing similar mutation into theexpression controlling region. Furthermore, it is possible to controlthe expressing time, expressing site, expression level and the like morepositively in combination with a Cre-loxP system. Examples of thismethod include a method in which a promoter which is expressed in aspecific region in the brain is used, and the objective gene is deletedonly in this region [Cell, 87, 7, 1317 (1996)] and a method in which theobjective gene is deleted organ-specifically at the intended stage usinga Cre-expressing adenovirus [Science, 278, 5335, (1997)].

[0436] Accordingly, expression of a DNA encoding the polypeptide of thepresent invention on the chromosome can also be controlled at any stageand in a tissue in this manner. Also, it is possible to prepare ananimal individual having any insertion, deletion or substitution in thetranslation region or expression controlling region.

[0437] Such an animal can induce symptoms of various diseases caused bythe polypeptide of the present invention at any stage at any degree inany region. Thus, the non-human knockout animal of the present inventionbecomes a markedly useful experimental animal for the treatment andprevention of various diseases caused by the polypeptide of the presentinvention. Particularly, it is markedly useful as an evaluation model oftherapeutic agents and preventive agents and physiologically functionalfoods, healthy foods and the like.

(11) Gene Therapy Agent which Comprises the DNA of the Present Inventionand an RNA Comprising a Sequence Homologous to the DNA

[0438] The gene therapy agent using a virus vector which comprises theDNA of the present invention and an RNA comprising a sequence homologousto the DNA can be produced by mixing the recombinant virus vectorsprepared in the (7) with a base material used in gene therapy agents[Nature Genet., 8, 42 (1994)]. The gene therapy agent using a virusvector which comprises the DNA of the present invention and an RNAcomprising a sequence homologous to the DNA can be used as a medicamentfor treating or preventing diseases which accompany changes in theexpression of the polypeptide of the present invention or of a DNAencoding the polypeptide such as inflammatory disease, cancer and tumormetastasis.

[0439] As the base material used in the gene therapy agents, any basematerial can be used, so long as it is a base material generally used ininjections. Examples include distilled water, a salt solution such assodium chloride or a mixture of sodium chloride and an inorganic salt, asugar solution such as mannitol, lactose, dextran or glucose, an aminoacid solution such as glycine or arginine, a mixed solution of anorganic acid solution or a salt solution with a glucose solution and thelike. Also, injections may be prepared according to the conventionalmethod as solutions, suspensions or dispersions using an auxiliary agentincluding an osmotic pressure controlling agent, a pH adjusting agent, aplant oil such as sesame oil or soybean oil, lecithin, and a surfactantsuch as a nonionic surfactant in these base materials. These injectionscan also be prepared as solid preparations for dissolving when used, bypowdering, freeze drying or the like. The gene therapy agent of thepresent invention can be used directly in the treatment when it is aliquid, or when it is a solid, by dissolving it just before the genetherapy in the above base material sterilized, if necessary. Theadministration method of the gene therapy agent of the present inventionincludes a topical administration method to effect its absorption intothe treating region of a patient.

[0440] A virus vector can be prepared by preparing a complex through thecombination of the DNA of the present invention having an appropriatesize with a polylysine-conjugate antibody specific for the adenovirushexon protein, and linking the thus obtained complex to an adenovirusvector. The virus vector stably reaches the target cell and isincorporated into the cell by endosome and degraded inside the cell, sothat it can express the gene efficiently.

[0441] As the RNA virus vectors other than retrovirus vectors, virusvectors based on Sendai virus as a (−) chain RNA virus have beendeveloped (Japanese Patent Application No. 9-517213, Japanese PatentApplication No. 9-517214), so that a Sendai virus vector into which theDNA of the present invention is inserted for the purpose of gene therapycan be prepared.

[0442] The DNA can also be transported into foci by a non-viral genetransfer method.

[0443] Examples of the non-viral gene transfer method well known in thefield include calcium phosphate coprecipitation method [Virology, 52,456-467 (1973); Science, 209, 1414-1422 (1980)], micro-injection methods[Proc. Natl. Acad. Sci. USA, 77, 5399-5403 (1980); Proc. Natl. Acad.Sci. USA, 77, 7380-7384 (1980); Cell, 27, 223-231 (1981); Nature, 294,92-94 (1981)], membrane fusion-mediated transfer methods via liposome[Proc. Natl. Acad. Sci. USA, 84, 7413-7417 (1987); Biochemistry, 28,9508-9514 (1989); J. Biol. Chem., 264, 12126-12129 (1989); Hum. GeneTher., 3, 267-275 (1992); Science, 249, 1285-1288 (1990); Circulation,83, 2007-2011 (1992)], direct DNA incorporation and receptor-mediatedDNA transfer methods [Science, 247, 1465-1468 (1990); J. Biol. Chem.,266, 14338-14342 (1991); Proc. Natl. Acad. Sci. USA, 87, 3655-3659(1991); J. Biol. Chem., 264, 16985-16987 (1989); BioTechniques, 11,474-485 (1991); Proc. Natl. Acad. Sci. USA, 87, 3410-3414 (1990); Proc.Natl. Acad. Sci. USA, 88, 4255-4259 (1991); Proc. Natl. Acad. Sci. USA,87, 4033-4037 (1990); Proc. Natl. Acad. Sci. USA, 88, 8850-8854 (1991);Hum. Gene Ther., 3, 147-154 (1991)] and the like.

[0444] Regarding the membrane fusion-mediated transfer method vialiposome, it has been reported based on a study on tumors that, topicalincorporation and expression of a gene in a tissue is possible bydirectly administering a liposome preparation to the tissue as thetarget [Hum. Gene Ther., 3, 399-410 (1992)]. Accordingly, similar effectis expected also in foci of diseases relating to the DNA and polypeptideof the present invention. A direct DNA incorporation is preferable fordirectly targeting the DNA at a focus. The receptor-mediated DNAtransfer is carried out, for example, by conjugating a DNA (in general,it has a form of covalently closed super-coiled plasmid) with a proteinligand via a polylysine. The ligand is selected based on the presence ofa corresponding ligand receptor on the cell surface of a target cell ortissue. The ligand-DNA conjugate can be injected directly to a bloodvessel as occasion demands and can be directed to a target tissue wherereceptor binding and inherence of DNA-protein complex occur. In order toprevent intracellular degradation of DNA, the endosome function can bedestroyed by simultaneous infection with adenovirus.

[0445] The above gene therapy agent which comprises the DNA of thepresent invention, an RNA comprising a sequence homologous to the DNA, arecombinant vector comprising the nucleic acid or the like is introducedinto cells and a polypeptide encoded by the DNA, differentiation isexpressed, so that mutual recognition, migration and the like of thecells can be controlled. Examples of the cells in this case includecorpuscles, nerve cells, stem cells cancer cells and the like.

[0446] In addition, differentiation of promyelocyte into granulocyte canbe accelerated by introducing the above gene therapy agent intopromyelocyte and expressing therein.

[0447] The present invention is specifically described in based onExamples. However, the examples are for descriptions and do not limitthe technical scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0448]FIG. 1 shows a result of the FACS analysis of the expression levelof poly-N-acetyllactosamine sugar chains in HCT-15 cells transfectedwith a G6 polypeptide expression plasmid.

[0449]FIG. 2 shows a result of the examination on the participation ofG6 polypeptide in the synthesis of a glycoprotein sugar chain orglycolipid sugar chain by treating HCT-15 cells transfected with a G6polypeptide expression plasmid with various sugar chain synthesisinhibitors and then analyzing the expression level of apoly-N-acetyllactosamine sugar chain by using FACS.

[0450]FIG. 3 shows a result of an experiment carried out by purifying asecreted FLAG peptide-fused G6 polypeptide produced in insect cells, andsubjecting it to SDS polyacrylamide gel electrophoresis and then tosilver staining. B shows a result of a test carried out by purifying asecreted FLAG peptide-fused G6 polypeptide produced in insect cells, andsubjecting it to SDS polyacrylamide gel electrophoresis and then toWestern blotting by using an anti-FLAG peptide antibody.

[0451]FIG. 4 shows a result of a test carried out by extracting neutralglycolipid from Namalwa cells transfected with a G6 polypeptideexpression plasmid, developing the extract on a TLC plate and thensubjecting it to orcinol staining. B shows a result of a test carriedout by extracting neutral glycolipid from Namalwa cells transfected witha G6 polypeptide expression plasmid, developing the extract on a TLCplate and then subjecting it to immunostaining by using ananti-N-acetyllactosamine antibody 1B2.

[0452]FIG. 5 shows a result of electrophoresis to examine the expressionlevels of G6 transcripts and β-actin transcripts in 36 human organs byusing competitive PCR. The histogram is a graph showing the expressionlevel of G6 transcripts when the expression level of β-actin is definedas 1,000.

BEST MODE FOR CARRYING OUT THE INVENTION

[0453] Unless otherwise indicated, the known methods described inMolecular Cloning, Second Edition were used as the genetic engineeringtechniques described as follows.

EXAMPLE 1 Cloning of β1,3-galactosyltransferase Homologue (Rat G6) Genefrom a Rat Tibia-derived cDNA Library

[0454] (1) Preparation of RNA

[0455] From rat tibia, 2.2 mg of total RNA was prepared by the guanidinethiocyanate-cesium trifluoroacetate method [Methods in Enzymology, 154,3 (1987)]. Next, 15.7 μg of mRNA was obtained as poly(A)⁺ RNA by passing2.0 mg of the total RNA through an oligo(dT) cellulose column(manufactured by Collaborative Research).

[0456] (2) Preparation of cDNA Library

[0457] Using 4.0 μg of the mRNA obtained in the above (1), synthesis ofcDNA, ligation of BamHI adapter and digestion with NotI were carried outaccording to the linker primer method [Preparation Methods of GeneLibrary (Idenshi Library no Sakusei-ho), edited by Hirosho Nojima,Yodo-sha, 1994]. A cDNA library in which 5′-terminal of cDNA is alwayspresent in the BamHI site side of the vector was constructed byinserting the thus obtained double-stranded cDNA between BamHI site andNotI site of a plasmid pBluescript II SK(−). As a host for cDNA libraryconstruction, Escherichia coli MC1061A [Molecular Cloning SecondEdition] was used.

[0458] (3) Random Sequence

[0459] Plasmid DNAs were obtained from each of the E. coli clonesobtained in the above (2) according to a conventional method, and 300-to 400-bp nucleotide sequences of the 5′-terminal and 3′-terminal sidesof cDNA contained in each plasmid were determined. The nucleotidesequence was determined by using a commercially available kit (DyeTerminator Cycle Sequencing FS Ready Reaction Kit, dRhodamine TerminatorCycle Sequencing FS Ready Reaction Kit or BigDye Terminator CycleSequencing FS Ready Reaction Kit, manufactured by PE Biosystems) and aDNA sequencer (ABI PRISM 377, manufactured by PE Biosystems). As theprimers, T3 primer (manufactured by STRATAGENE) and T7 primer(manufactured by STRATAGENE) were used.

[0460] Genes and proteins having homology were analyzed by using theprograms of BLAST [J. Mol. Biol., 215, 403-410 (1990)] for the thusobtained nucleotide sequences, or FrameSearch [manufactured by Compugen]for amino acid sequences deduced from the nucleotide sequences. As aresult, it was considered that a cDNA contained in a plasmid namedOVX2-038 encodes a protein having homology with aβ1,3-galactosyltransferase β3Gal-T1 (alias WM1: Japanese PublishedUnexamined Patent Application No. 181759/94). A nucleotide sequence (738bp) of the cDNA contained in OVX2-038 is shown in SEQ ID NO:3, and anamino acid sequence of a polypeptide considered to be encoded by the DNAis shown in SEQ ID NO:32. It was considered that the cDNA contained inOVX2-038 is a partial fragment of a cDNA encoding a ratβ1,3-galactosyltransferase homologue (named rat G6).

EXAMPLE 2 Search of Gene Encoding Human G6

[0461] Human G6 gene corresponding to the rat G6 cDNA obtained inExample 1 was searched. As a result of the search of genes havinghomology with the nucleotide sequence (represented by SEQ ID NO:3) ofthe cDNA contained in OVX2-038 obtained in Example 1, or genes having apossibility to encode polypeptides having homology with the polypeptide(represented by SEQ ID NO:32) considered to be encoded by the cDNA atamino acid level from gene data bases by using the programs of BLAST [J.Mol. Biol., 215, 403-410 (1990)] and FrameSearch [manufactured byCompugen], one EST (expressed sequence tag) sequence (GenBank No.AI039637) was found. Since the EST sequence is 428 bp, a total aminoacid sequence of a polypeptide encoded by a gene corresponding to thisEST and the function of the polypeptide cannot be found from thissequence information alone.

EXAMPLE 3 Cloning of Human G6 cDNA

[0462] Cloning of candidate gene fragments was attempted by designing aprimer set specific for the EST sequence (GenBank No. AI03937) found inExample 2. As the primers, CB-462 having the nucleotide sequencerepresented by SEQ ID NO:4 and CB-464 having the nucleotide sequencerepresented by SEQ ID NO:5 were used. Using the primer set, the presenceof human G6 cDNA was examined by PCR by using single-stranded cDNAsprepared from various organs, or various cDNA libraries, as templates.As a result, a DNA fragment of about 250 bp was amplified when a cDNAlibrary derived from a human colon cancer cell line Colo205 or a cDNAlibrary derived from human gastric mucosa was used as a template.

[0463] A human G6 cDNA clone of about 3 kb was obtained by screening theabove two cDNA libraries by using the amplified fragment as a probe.Since the clone was lacking the 5′-terminal portion, a 5′-terminal sideDNA of the human G6 cDNA was obtained by using 5′ RACE method.Full-length sequence of the human G6 cDNA (SEQ ID NO:2) was determinedby sequencing the cDNA clone of about 3 kb of human G6 cDNA and the5′-terminal side DNA, and connecting these sequences. A DNA fragmentcontaining entire coding regions was obtained by PCR by using primersconstructed based on the sequence. The absence of mutation in thenucleotide sequence derived by PCR was confirmed by sequencing thefragment. Specific methods are shown below.

[0464] (1) Preparation of a cDNA Library Derived from Human Colon CancerCell Line Colo205 and Analysis by PCR

[0465] About 30 μg of mRNA was obtained from the human colon cancer cellline Colo205 by using a mRNA extraction kit Oligotex™-dT30 <super>(manufactured by Roche). Specific reagents and method are as describedin the instructions attached to the kit. Using 8 μg of the thus obtainedmRNA and SUPERSCRIPT Choice System for cDNA Synthesis Kit (manufacturedby GIBCO BRL), double-stranded cDNAs were synthesized using oligo(dT) asa primer.

[0466] An SfiI linker was ligated to both ends of the double-strandedcDNAs by the following method. A single-stranded DNA (11 nucleotides)having the nucleotide sequence represented by SEQ ID NO:6 and asingle-stranded DNA (8 nucleotides) having the nucleotide sequencerepresented by SEQ ID NO:7, which constitute the SfiI linker weresynthesized by using 380A DNA synthesizer (manufactured by AppliedBiosystems).

[0467] Fifty micrograms of the respective synthesized single-strandedDNAs at 50 μg were separately dissolved in 50 μl of a buffer containing50 mmol/l Tris-HCl (pH 7.5), 10 mmol/l MgCl₂, 5 mmol/l dithiothreitol(hereinafter referred to as “DTT”), 0.1 mmol/l EDTA(ethylenediaminetetraacetic acid) and 1 mmol/l ATP (hereinafter referredto as “T4 kinase buffer”), and the 5′-end was phosphorylated by adding30 units of T4 polynucleotide kinase (manufactured by Takara Shuzo) andcarrying out the phosphorylation reaction at 37° C. for 16 hours.

[0468] In 45 μl of the T4 ligase buffer, 4 μg of the synthetic DNA of 11nucleotides and 2.9 μg of synthetic DNA of 8 nucleotides whose 5′-endswere phosphorylated, and the double-stranded cDNAs synthesized in theabove were dissolved, and then 1,050 units of T4 DNA ligase was addedthereto for reaction at 16° C. for 16 hours to thereby ligate the SfiIlinker to each of the double-stranded cDNAs.

[0469] DNA fragments of about 1.5 kb or more were recovered bysubjecting the thus obtained reaction solution to agarose gelelectrophoresis.

[0470] In 590 μl of a buffer solution comprising 10 mmol/l Tris-HCl (pH7.5), 6 mmol/l MgCl₂, 50 mmol/l NaCl and 6 mmol/l 2-mercaptoethanol(hereinafter referred to as “Y-50 buffer”), 24 μg of an expressioncloning vector pAMo [J. Biol. Chem., 268, 22782 (1003), alias pAMoPRC3Sc(Japanese Published Unexamined Patent Application No. 336963/93)] wasdissolved, and then 80 units of SfiI (manufactured by Takara Shuzo,hereinafter, unless otherwise indicated, restriction enzymesmanufactured by Takara Shuzo were used) was added thereto for digestionat 37° C. for 16 hours.

[0471] To the reaction solution, 40 units of BamHI were added, and thedigestion was carried out 37° C. for 2 hours. A DNA fragment of about8.8 kb was recovered by subjecting the reaction solution to agarose gelelectrophoresis.

[0472] After each of the SfiI linker-added double-stranded cDNAsprepared in the above (derived from 8 μg of mRNA) was dissolved in 250μl of the T4 ligase buffer, 2 μg of the DNA fragment of about 8.8 kb and2,000 units of T4 DNA ligase were added to each of the solutions, andthe ligation reaction was carried out at 16° C. for 16 hours.

[0473] After the reaction, 5 μg of transfer RNA (tRNA) was added to eachof the reaction solutions and subjected to ethanol precipitation, andthe precipitate was dissolved in 200 μl of a buffer comprising 10 mmol/lTris-HCl (pH 8.0) and 1 mmol/l EDTA (hereinafter referred to as “TEbuffer”).

[0474] Using the reaction solution, E. coli LE392 (Molecular Cloning,Second Edition) was transformed by electroporation [Nucleic Acids Res.,16, 6127 (1988)] to give about one million transformants havingampicillin resistance, and thus a cDNA library was constructed.

[0475] Subsequently, cDNA-containing plasmids were prepared using thecDNA library (E. coli) and a plasmid preparation kit /plasmid/maxi kit(manufactured by QIAGEN, product No. 41031).

[0476] The presence of human G6 cDNA was examined by PCR using theplasmid DNAs as templates and CB-462 having the nucleotide sequencerepresented by SEQ ID NO:4 and CB-464 having the nucleotide sequencerepresented by SEQ ID NO:5 as primers. The PCR was carried out under thefollowing conditions. A reaction solution (50 μl) comprising 10 ng/ml ofthe plasmid DNA, 10 mmol/l Tris-HCl (pH 8.3), 50 mmol/l KCl, 1.5 mmol/lMgCl₂, 0.2 mmol/l dNTP, 0.001% (w/v) of gelatin, 0.2 μmol/l human G6gene specific primers (CB-462 and CB-464) and 1 unit of AmpliTaq GoldDNA polymerase (manufactured by Perkin Elmer) was heated at 95° C. for11 minutes, and then 50 cycles of the reaction was carried out, eachcycle consisting of 30 seconds at 95° C., 1 minute at 55° C. and 2minutes at 72° C. As a result of the PCR, a DNA fragment of about 250 bpconsidered to be derived from human G6 cDNA was amplified from severalpools.

[0477] (2) Production of a Human Gastric Mucosa cDNA Library

[0478] A human gastric mucosa cDNA library was produced as follows. AcDNA was synthesized from human gastric mucosa poly(A)+RNA using cDNASynthesis System (manufactured by GIBCO BRL), and after adding anEcoRI-NotI-SalI adapter (Super Choice System for cDNA Synthesis;manufactured by GIBCO BRL) to both ends thereof, inserted into the EcoRIsite of a cloning vector λZAP II (λZAP II/EcoRI/CIAP Cloning Kit,manufactured by STRATAGENE) and then subjected to in vitro packagingusing Gigapack III Gold Packaging Extract (manufactured by STRATAGENE)to thereby produce a cDNA library.

[0479] The gastric mucosa cDNA library (phage library) was divided intopools each containing about 50,000 independent clones, and then PCR wascarried out using the phage (about 1×10⁷ particles) of each pool as atemplate. The method was similar to the method described in the above(1), except that the phage (about 1×10⁷ particles) heat-treated at 99°C. for 10 minutes was used as a template. As a result of the PCR, a DNAfragment of about 250 bp considered to be derived from human G6 cDNA wasamplified from several pools. The DNA fragment was purified usingPrep-A-Gene DNA Purification Kit (manufactured by BIO RAD) and digestedwith a restriction enzyme (AvaII or RsaI) to confirm that the amplifiedfragment has a partial sequence (the sequence of positions 946 to 1196of SEQ ID NO:2, 251 bp) of the above EST sequence (GenBank No.AI039637). The amplified fragment was digested into 3 DNA fragments of96 bp, 80 bp and 72 bp by AvaII digestion, and into 2 DNA fragments of176 bp and 72 bp by RsaI digestion.

[0480] (3) Cloning of Human G6 cDNA

[0481] Using Multiprime DNA Labelling System (manufactured by AmershamPharmacia Biotech), the PCR-amplified DNA fragment of about 250 bpobtained in the above (2) was labeled with a radioisotope to prepare aprobe. The DNA fragment (50 ng) and 50 μl of a reaction solutioncontaining a random primer and radioisotope ([α-³²P]dCTP) were allowedto react at 37° C. for 30 minutes. The composition of the reactionsolution and the handling are as described in the manufacture'sinstructions attached to the kit. Next, the reaction was stopped byvoltex, and the radioisotope-labeled probe was purified by gelfiltration using a Sephadex G50 column.

[0482] Plaque hybridization was carried out by using theradioisotope-labeled probe on 7 pools (a total of about 350,000independent clones) of the gastric mucosa cDNA library in whichamplification was found in the above (2).

[0483] Filters (Biodine A: manufactured by PALL) on which plaque-derivedDNAs were transferred were soaked in 25 ml of a buffer comprising5-folds concentration SSPE [composition of 1-fold concentration SSPEcomprises 180 mmol/l sodium chloride, 10 mmol/l sodiumdihydrogenphosphate and 1 mmol/l EDTA (pH 7.4)], 5-folds concentrationDenhardt solution [composition of 1-fold Denhardt solution comprises0.02% (w/v) bovine serum albumin, 0.02% (w/v) Ficoll 400 and 0.02% (w/v)polyvinyl pyrrolidone], 0.5% sodium dodecyl sulfate (SDS) and 20 μg/mlsalmon sperm DNA (hereinafter referred to as “hybridization buffer”),and pre-hybridization was carried out at 65° C. for 1 hour.

[0484] Next, the filters were soaked in 10 ml of the hybridizationbuffer containing the radioisotope-labeled probe prepared in the above,and hybridization was carried out at 65° C. for 16 hours.

[0485] Thereafter, the filters was washed twice under conditions ofsoaking it at 42° C. for 20 minutes in a buffer solution comprising0.1-fold concentration SSC [composition of 1-fold concentration SSCcomprises 15 mmol/l sodium citrate and 150 mmol/l sodium chloride (pH7.0)] and 0.1% SDS.

[0486] As a result of the plaque hybridization, 11 hybridizingindependent phage clones were obtained. Each phage clone was convertedinto a plasmid clone by carrying out in vivo excision using a kitmanufactured by STRATAGENE. Thus, a plasmid in which an insert cDNA ofeach phage clone is inserted into pBluescript SK(−) can be obtained. Themethod followed the manufacture's instructions attached to the kit.

[0487] The size of cDNA contained in each plasmid was examined bydigesting the thus obtained 11 plasmids with a restriction enzyme EcoRI.As a result, it was found that all plasmids contain a cDNA of about 3kb. From the digestion patterns with plural restriction enzymes (PstI,HindIII and BsmI), it was considered that all of the 11 plasmids areidentical. One of these plasmids was named pBS-G6s.

[0488] (4) Determination of Nucleotide Sequence of cDNA Inserted intoPlasmid pBS-G6s

[0489] A full nucleotide sequence of the cDNA contained in pBS-G6s wasdetermined by the following method.

[0490] Using primers specific for a sequence in pBluescript SK(−)[M13(-20) Primer and M13 Reverse Primer: manufactured by TOYOBO],5′-terminal and 3′-terminal sequences of the cDNA were determined.Synthetic DNAs specific for the determined sequences were prepared andused as primers, and further continuing nucleotide sequences weredetermined. A complete nucleotide sequence of the cDNA was determined byrepeating the process.

[0491] A DNA sequencer Model 4000L (manufactured by LI-COR) and areaction kit (Sequitherm EXCELL II™ Long-Read™ DNA-sequencing kit-Lc:manufactured by Air Brown) or a DNA sequencer 377 (manufactured byPerkin Elmer) and a reaction kit (ABI Prism™ BigDye™ Terminator CycleSequencing Ready Reaction Kit: manufactured by Applied Biosystems) wereused for the determination of the nucleotide sequence.

[0492] It was revealed that the cDNA contained in pBS-G6s has anucleotide sequence (total nucleotide sequence 2,762 bp) of positions289 to 3,750 in SEQ ID NO:2. It was considered, based on the nucleotidesequence, that the cDNA encodes a human β1,3-galactosyltransferasehomologue (human G6), but it was lacking an N-terminal side polypeptideregion.

[0493] (5) Cloning of Full-length cDNA of Human G6

[0494] From the result of the above (1), it was found that a human G6transcript is expressed in a colon cancer cell line Colo205.Accordingly, 5′ RACE method using total RNA of Colo205 as the templatewas carried out to obtain a 5′-terminal DNA fragment of human G6 cDNA.The 5′ RACE method was carried out using a kit (5′-RACE Systems forRapid Amplification of cDNA Ends, Version 2; manufactured by LifeTechnologies).

[0495] As G6 cDNA specific primers, synthetic DNAs having the nucleotidesequences shown in SEQ ID NOs:8 and 9 were used. As a result, a DNAfragment of about 460 bp was amplified. The DNA fragment was blunt-endedaccording to a conventional method and then inserted into the EcoRV siteof pBluescript SK (−). Next, the cDNA was sequenced using primersspecific for a sequence in pBluescript SK(−) [M13(−20) Primer and M13Reverse Primer: manufactured by TOYOBO]. A DNA sequencer Model 4000L(manufactured by LI-COR) or a DNA sequencer 377 (manufactured by PerkinElmer) and a reaction kit for each sequencer were used for sequencing.The DNA fragment (named RO2-16) has a nucleotide sequence (totalnucleotide sequence 457 bp) of positions 513 to 969 in SEQ ID NO:2. TheDNA fragment was unable to cover the N-terminal region of the G6polypeptide. Accordingly, the following test was carried out.

[0496] A 5′-terminal side DNA fragment of the human G6 cDNA can beamplified by PCR using the gastric mucosa cDNA library constructed inthe above (2) as a template, and a primer specific for the vector and aprimer specific for the human G6 cDNA. Specifically, M13 Reverse Primer(manufactured by TOYOBO) which is a primer specific for a sequence inpBluescript SK(−) was used as a primer specific for the vector, and asynthetic DNA having the nucleotide sequence represented by SEQ ID NO:10as the primer specific for human G6 cDNA. The PCR was carried out using50 ng/ml of the cDNA library (plasmid) as a template under theconditions described in the above (1). Next, PCR was carried out using 1μl of the PCR solution as a template, and M13 Reverse Primer(manufactured by TOYOBO) as a primer specific for a sequence inpBluescript SK(−) and a synthetic DNA having the nucleotide sequencerepresented by SEQ ID NO:9 as a primer specific for the human G6 cDNA.The PCR was carried out under the conditions described in the above (1).As a result, a DNA fragment of about 1 kb was amplified.

[0497] The DNA fragment was blunt-ended according to a conventionalmethod and then inserted into the EcoRV site of pBluescript SK (−).Next, the DNA fragment was sequenced using a primer specific for asequence in pBluescript SK(−) [M13(-20) Primer or M13 Reverse Primer:both manufactured by TOYOBO]. A synthetic DNA specific for thedetermined sequence was prepared and used as a primer, and furthercontinuing nucleotide sequences were determined. A complete nucleotidesequence of the DNA fragment was determined by repeating the process. ADNA sequencer Model 4000L (manufactured by LI-COR) or a DNA sequencer377 (manufactured by Perkin Elmer) and a reaction kit for each sequencerwere used for sequencing. The DNA fragment (named No. 19) has anucleotide sequence (total nucleotide sequence 969 bp) of positions 1 to969 in SEQ ID NO:2.

[0498] A nucleotide sequence of cDNA encoding the full-length G6polypeptide was determined by connecting the thus determined sequence ofcDNA contained in pBS-G6s, the sequence of PCR fragment determined inthe above (2), the sequence of RO2-16 and the sequence of No. 19. Thethus determined nucleotide sequence of human G6 full-length cDNA (3,750bp) is shown in SEQ ID NO:2. The cDNA encoded a polypeptide comprising378 amino acids having structure characteristic to glycosyltransferase.This polypeptide is called G6 polypeptide, and its amino acid sequenceis shown in SEQ ID NO:1.

[0499] In order to obtain a DNA encoding the full-length G6 polypeptide,PCR was carried out using the cDNA library of human colon cancer cellline Colo205 as a template and primers specific for the human G6 cDNA.Specifically, PCR was carried out by heating 50 μl of a reactionsolution containing 50 ng/ml of the cDNA library (plasmid), primersincluding an EcoRI recognition sequence [CB-497 (SEQ ID NO:11) andCB-501 (SEQ ID NO:12)] and Platinum Pfx DNA polymerase (manufactured byGIBCO BRL) (detailed composition is described in the Platinum Pfx DNApolymerase kit) at 94° C. for 2 minutes and then carrying out 45 cyclesof the reaction, each cycle consisting of 20 seconds at 94° C., 45seconds at 55° C. and 2 minutes at 68° C. As a result, a DNA fragment ofabout 1.3 kb was amplified. The fragment was digested with EcoRI andthen inserted into the EcoRI site of pBluescript SK(−) to obtain aplasmid pBS-G6. As a result of sequencing the inserted DNA fragment, itwas confirmed that the DNA fragment has a nucleotide sequence ofposition 46 to 1,372 in SEQ ID NO:2. A DNA sequencer 377 (manufacturedby Perkin Elmer), a reaction kit for the sequencer and human G6cDNA-specific primers were used for sequencing. The DNA fragment encodeda polypeptide comprising 378 amino acids having structure characteristicto glycosyltransferase. This polypeptide was possessed of the same aminoacid sequence of G6 polypeptide. The absence of errors in the sequenceof the PCR-amplified DNA fragment inserted into pBS-G6 was confirmed bycarrying out direct sequencing using the PCR-amplified DNA fragment ofabout 1.3 kb.

[0500] A nucleotide sequence of cDNA encoding the full-length G6polypeptide was determined by connecting the thus determined sequence ofcDNA contained in pBS-G6s, the sequence of PCR fragment determined inthe above (2), the sequence of No. 19 and the sequence of PCR-amplifiedDNA fragment contained in pBS-G6. The thus determined nucleotidesequence of human G6 full-length cDNA (3,750 bp) was the same as thenucleotide sequence of SEQ ID NO:2.

EXAMPLE4 Homology Analysis

[0501] The G6 polypeptide showed homologies of 35.6%, 32.8%, 30.8%,30.3% and 33.9% at amino acid level with so far cloned five humanβ1,3-galactosyltransferases β3Gal-T1 (Japanese Published UnexaminedPatent Application No. 181759/94), β3Gal-T2 [J. Biol. Chem., 273,433-440 (1998), J. Biol. Chem., 273, 12770-12778 (1998)], β3Gal-T3 [J.Biol. Chem., 273, 12770-12778 (1998)], β3Gal-T4 [J. Biol. Chem., 273,12770-12778 (1998)) and β3Gal-T5 [J. Biol. Chem., 274, 12499-12507(1999)], respectively. Also, the polypeptide showed a homology of 29.4%at amino acid level with so far cloned humanβ1,3-N-acetylglucosaminyltransferase (β3GnT1) [Proc. Natl. Acad. Sci.USA, 96, 406-411 (1999)].

[0502] It is considered from the amino acid sequence (SEQ ID NO:1) thatthe G6 polypeptide is a type II membrane protein characteristic toglycosyltransferase. It was considered that it comprises an N-terminalcytoplasmic region containing 14 amino acids, subsequentmembrane-binding region containing 18 amino acids rich in hydrophobicproperty, a stem region containing at least 12 amino acids and theremaining most part of the C-terminal region containing a catalyticregion. Based on the comparison of amino acid sequences with the aboveglycosyltransferases having a homology and information on stem regionsand catalytic regions of the above glycosyltransferases (JapanesePublished Unexamined Patent Application No. 181759/94), it was assumedthat the stem region contains at least 12 amino acids. Accordingly, itis considered that the polypeptide comprising an amino acid sequence ofpositions 45 to 378 contains a catalytic region.

[0503] Based on these results and the results of Examples 6, 7, 9 and 10which are described below, it was found that the polypeptide is a novelβ1,3-N-acetylglucosaminyltransferase and the polypeptides comprisingamino acid sequences of positions 36 to 378 in SEQ ID NO:1 and 39 to 378in SEQ ID NO:1 are secretory polypeptides.

EXAMPLES5 Construction of Expression Plasmids for Animal Cells

[0504] In order to express the G6 polypeptide encoded by the human G6cDNA obtained in Example 3 in animal cells, an expression plasmid wasconstructed by introducing human G6 cDNA into an expression plasmid pAMo[J. Biol. Chem., 266, 22782 (1993), alias pAMoPRC3Sc (Japanese PublishedUnexamined Patent Application No. 336963/93)] or pCXN2 [Gene, 108, 193(1991)].

[0505] (1) Construction of a Plasmid pAMo-G6 for Expressing G6Polypeptide

[0506] The pBS-G6 was digested with a restriction enzyme EcoRI and thenconverted to be blunt-ended by using a DNA polymerase Klenow fragment.Thereafter, a SfiI fragment of about 1,350 bp was obtained by adding theSfiI linker prepared in Example 3(1). Separately, pAMo was digested withSfiI and BamHI and then an SfiI fragment of 8.7 kb was obtained. Anexpression plasmid pAMo-G6 was constructed by ligating the twofragments.

(2) Construction of a Plasmid pCXN2-G6 for Expressing G6 Polypeptide

[0507] The pBS-G6 was digested with a restriction enzyme EcoRI and thenan EcoRI fragment of about 1,330 bp was obtained. Separately, pCXN2 wasdigested with EcoRI and then an EcoRI fragment of about 3 kb wasobtained. An expression plasmid pCXN2-G6 was constructed by ligating thetwo fragments.

EXAMPLE 6 Synthesis of a Poly-N-acetyllactosamine Sugar Chain in HumanCulture Cells Transfected with a G6 Polypeptide Expression Plasmid

[0508] (1) Preparation of Stable Transformant Using Namalwa Cell as aHost

[0509] Each of a control plasmid (pAMo) and the G6 polypeptideexpression plasmid (pAMo-G6) constructed in Example 5 was dissolved inTE buffer to give a concentration of 1 μg/μl and then transfected into ahuman B cell line Namalwa cell by electroporation [Cytotechnology, 3,133 (1990)] to obtain transformed cells.

[0510] After the plasmids were transfected at 4 μg per 1.6×10⁶ cells,the cells were suspended in 8 ml of 10% fetal bovine serum-containingRPMI 1640 medium [RPMI 1640 medium (manufactured by NissuiPharmaceutical) supplemented with 1/40 volume of 7.5% NaHCO₃, 3% 200mmol/l L-glutamine solution (manufactured by GIBCO) and 0.5%penicillin-streptomycin solution (manufactured by GIBCO, 5,000 units/mlpenicillin and 5,000 μg/ml streptomycin); hereinafter, RPMI 1640 mediummeans the RPMI 1640 medium supplemented with these additives] andcultured at 37° C. for 24 hours in a CO₂ incubator. After the culturing,G418 (manufactured by GIBCO) was added thereto to give a concentrationof 0.8 mg/ml, followed by culturing for 14 days to obtain a stabletransformant. The transformant was sub-cultured using RPMI 1640 mediumcontaining 0.8 mg/ml G418 and 10% fetal bovine serum.

[0511] (2) Preparation of a Stable Transformant Using HCT-15 Cell as aHost

[0512] Each of a control plasmid (pCXN2) and the G6 polypeptideexpression plasmid (pCXN2-G6) constructed in Example 5 was dissolved inTE buffer to give a concentration of 1 μg/μl and then transfected into ahuman colon cancer cell line HCT-15 cell by the electroporation method[Cytotechnology, 3, 133 (1990)] to obtain transformed cells.

[0513] After the plasmids were transfected at 10 μg per 8×10⁶ cells, thecells were suspended in 8 ml of 10% fetal bovine serum-containing RPMI1640 medium and cultured at 37° for 24 hours in a CO₂ incubator.

[0514] After the culturing, G418 (manufactured by GIBCO) was addedthereto to a concentration of 0.8 mg/ml, and the culturing was continuedfor 20 days to obtain a stable transformant. In addition, a single clonewas also obtained from the transformed cells using limiting dilution.The transformant was sub-cultured using RPMI 1640 medium containing 0.8mg/ml of G418 and 10% of fetal bovine serum.

[0515] (3) Measurement of Expression Level of Poly-N-acetyllactosamineSugar Chains in Respective Transformed Cells

[0516] An example by using an anti-i antibody (OSK28) which recognizespoly-N-acetyllactosamine sugar chains is shown below.

[0517] The HCT-15 cells transfected with pCXN2-G6 or pCXN2 prepared inthe above (2) (each 5×10⁶ cells) were washed by using 3 ml of aphosphate buffer PBS (8 g/l NaCl, 0.2 g/l KCl, 1.15 g/l Na₂HPO₄(anhydrous) and 0.2 g/l KH₂PO₄).

[0518] The cells (about 1×10⁶ cells) described above were put into amicrotube (1.5 ml, manufactured by Eppendorf) and the cells werecollected by centrifugation (550× g, 7 minutes).

[0519] The cells were washed with 0.9 ml of 0.1% sodium azide-containingPBS (A-PBS: 8 g/l NaCl, 0.2 g/l KCl, 1.15 g/l Na₂HPO₄ (anhydrous), 0.2g/l KH₂PO₄ and 0.1% sodium azide), and then the washed cells weresuspended in 100 μl of an antibody (OSK28) capable of recognizing apoly-N-acetyllactosamine sugar chain diluted with A-PBS to give aconcentration of 10 μg/ml (20 times dilution of the following purifiedantibody) and allowed to react at 4° C. for 30 minutes in the dark.

[0520] OSK28 (a purified antibody) was obtained from Dr. Junko Takahashiat the First Research Department, Osaka Red Cross Blood Center. OSK28 isa human monoclonal antibody (IgM antibody) produced by an immortalizedB-cell line established from human lymphocytes capable of producing ananti-Tja+anti-i antibodies by an EBV-hybridoma method. The purifiedOSK28 used in this test was purified by the method described in the BestMode for Carrying Out the Invention (6)(i) after a large scale culturingof the above B-cell line.

[0521] After the reaction, the cells are washed twice with 3 ml ofA-PBS, suspended in 100 μl of an FITC-labeled anti-human IgM antibody[manufactured by Medical & Biological Laboratories (MBL)] diluted to 10μg/ml, and then allowed to react at 4° C. for 1 hour. After thereaction, the cells were washed twice with 3 ml of A-PBS, suspended in200 μl of PBS containing 0.5% p-formaldehyde and then immobilized. Thecells were analyzed by using FACS (Epics Elite Flow Cytometer). Also, asa control test, the same analysis was carried out by using A-PBS insteadof the antibody.

[0522] HCT-15 cells transfected with the G6 polypeptide expressionplasmid (pCXN2-G6) or the control plasmid pCXN2 were subjected toindirect fluorescent antibody staining by using various anti-sugar chainantibodies (OSK28, KM93, OM81, CA19-9, KM231, TT42 and 7LE) and thenanalyzed by using FACS, and the results are shown in FIG. 1. The FACSanalysis using indirect fluorescent antibody staining was carried outaccording to a conventional method [J. Biol. Chem., 274, 12499-12507(1999)].

[0523] The reactivity to OSK28 was increased in the cells transfectedwith pCXN2-G6 in comparison with the cells transfected with pCXN2 (FIG.1). These results mean that a poly-N-acetyllactosamine sugar chain wasnewly synthesized on sugar chains of a glycoprotein or glycolipid on thesurface of cells by expressing the G6 polypeptide in HCT-15 cells.

[0524] On the other hand, when the fluorescent staining was carried outusing CA19-9 or KM231 as an antibody against a sialyl Lewis a sugarchain, the reactivity to the antibody did not change in HCT-15 cellstransfected with pCXN2-G6 and HCT-15 cells transfected with pCXN2 (FIG.1). It is known that HCT-15 cells express αl,3/1,4-fucose transferase(Fuc-TIII), and a sialyl Lewis a sugar chain which is detected by theabove antibody is synthesized when β1,3-galactosyltransferase isexpressed in the cells [J. Biol. Chem., 274, 12499-12507 (1999)]. Thus,the above results show that the G6 polypeptide does not have the GlcNAcβ1,3-galactosyltransferase activity.

[0525] In the same manner, when the fluorescent staining was carried outby using KM93 as an antibody against a sialyl Lewis x sugar chain, PM81as an antibody against a Lewis x sugar chain, TT42 as an antibodyagainst a Lewis b sugar chain or 7LE as an antibody against a Lewis asugar chain, the reactivity to the antibody also did not change inHCT-15 cells transfected with pCXN2-G6 and HCT-15 cells transfected withpCXN2 (FIG. 1).

[0526] (4) Experiments Using Sugar Chain Synthesis Inhibitors

[0527] Experiments using sugar chain synthesis inhibitors were carriedout in order to examine whether the G6 polypeptide is involved in thesynthesis of a glycoprotein sugar chain or a glycolipid sugar chain. ThepCXN2-G6-transfected HCT-15 cell (single clone) obtained in the above(2) was cultured for 5 days in the presence of various sugar chainsynthesis inhibitors, and then analysis by FACS was carried out usingOSK28. An inhibitor for O-linked sugar chain of glycoproteins,benzyl-α-GalNAc (manufactured by SIGMA) was used at a concentration of 4mmol/l. As an inhibitor for N-linked sugar chain of glycoproteins, amannosidase II inhibitor swainsonine (manufactured by SeikagakuCorporation) was used at a concentration of 10 μg/ml. As a glycolipidsugar chain inhibitor, a glucosylceramide synthase inhibitor D-PDMP(D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol: manufacturedby Matreya) was used at a concentration of 10 μmol/l. Also, as anegative control of D-PDMP, L-D-PDMP(L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol: manufacturedby Matreya) was used at a concentration of 10 μmol/l. The culturing wascarried out according to the method of the above (2). The results areshown in FIG. 2. As a control, the indirect fluorescent antibodystaining using OSK28 was carried out on HCT-15 cells transfected with avector (pCXN2), and the result is also shown in the drawing.

[0528] When cultured in the presence of a sugar chain synthesisinhibitor benzyl-α-GalNAc, the reactivity to OSK28 antibody was sharplyreduced in comparison with the case of its absence. This result suggeststhat the de novo synthesis poly-N-acetyllactosamine sugar chains of G6polypeptide-expressed observed in HCT-15 cells is occurred mainly on theO-linked sugar chains of glycoproteins. On the other hand, since thereactivity to OSK28 antibody was also reduced in swainsonine-treatedcells, it suggests that the de novo synthesis of apoly-N-acetyllactosamine sugar chains observed in G6polypeptide-expressed HCT-15 cells is also occurred on N-linked sugarchains of glycoproteins. Based on the above results, it is consideredthat when the G6 polypeptide is highly expressed in animal cells, apoly-N-acetyllactosamine sugar chain can be synthesized on glycoproteinsugar chains.

[0529] Furthermore, it is considered that a poly-N-acetyllactosaminesugar chain is newly synthesized also on sugar chains of glycoproteinsand oligosaccharides secreted from cells in which the G6 polypeptide isexpressed. Accordingly, useful secreted glycoproteins are produced byusing a G6 polypeptide-expressed cell as a host, so that sugar chainscontaining a poly-N-acetyllactosamine sugar chain can be added to thethus produced secretory glycoproteins.

EXAMPLE 7 Measurement of β1,3-N-acetylglucosaminyltransferase Activityin Human Cultural Cells Transfected with a G6 Polypeptide ExpressionPlasmid

[0530] Using a cell extract of the stable transformant cell (Namalwacell) transfected with the G6 polypeptide expression -plasmid obtainedin Example 6(1), β1,3-N-acetylglucosaminyltransferase activity wasmeasured.

[0531] (1) Activity Measurement by Using 2-aminobenzamide-labeledOligosaccharides as Substrates

[0532] The transformant cells (about 2×10⁷ cells) obtained in Example6(1) were put into a microtube (1.5 ml: manufactured by Eppendorf), andthe cells were collected by centrifugation (550×g, 7 minutes). The cellswere washed with 0.9 ml of PBS, the washed cells were suspended in asolution (100 μl) containing 20 mmol/l HEPES (pH 7.2) and 2% TritonX-100 and then the cells were disrupted using a sonicator (Bioruptor;manufactured by COSMO BIO). After the mixture was allowed to stand at 4°C. for 1 hour, the supernatant was obtained by centrifugation (550×g, 7minutes). The supernatant was used as an enzyme sample.

[0533] A β1,3-N-acetylglucosaminyltransferase activity was measured byusing this enzyme sample and 2-aminobenzamide-labeled sugar chainsubstrates.

[0534] The 2-aminobenzamide-labeled sugar chain substrates were preparedby using SIGMA 2AB glycan labeling kit (manufactured by OxfordGlycoscience) according to the manufacture's instructions of the kit.2-Aminobenzamide-labeled lacto-N-neotetraose(Galβ1,4-GlcNAcβ1-3Galβ1-4Glc; hereinafter referred to as “LNnT”) andGalβ1,4-GlcNAcβ1-3Galβ1-4GlcNAc (hereinafter referred sometimes to as“2LN”) were used as substrates. LNnT was purchased from OxfordGlycosystems. 2LN was obtained from Seikagaku Corporation.

[0535] The activity measurement was carried out using known methods[FEBS, 462, 289 (1999), J. Biol. Chem., 269, 14730-14737 (1994), J.Biol. Chem., 267, 23507 (1992), J. Biol. Chem., 267, 2994 (1992)].Specifically, the reaction was carried out at 37° C. for 16 hours in 20μl of an assay solution [150 mmol/l MOPS (pH 7.5), 50 mmol/l UDP-GlcNAc(manufactured by SIGMA), 20 mmol/l sodium cacodylate (pH 7.2), 0.4%Triton CF-54, 10 mmol/l MnCl₂, 15 mmol/l 2-aminobenzamide-labeled sugarchain substrate, the above cell extract (20 μg as protein)], and thenthe product was detected by high performance liquid chromatography(HPLC). The protein concentration of the cell extract was measured byusing DC Protein Assay Kit (manufactured by BIO RAD) according to themanufacture's instructions of the kit.

[0536] After carrying out the reaction by using an assay solutioncontaining UDP-GlcNAc (saccharide donor) and an assay solutioncontaining no donor and subsequently analyzing by HPLC, peaks appearedonly in the assay solution containing UDP-GlcNAc was defined asproducts.

[0537] The assay solution after completion of the reaction was treatedat 100° C. for 5 minutes, 50 μl of pure water for HPLC was addedthereto, and the mixture was centrifuged at 10,000×g for 5 minutes toobtain the supernatant. To a tube containing the assay solution, 50 μlof pure water for HPLC was again added to wash the tube, the mixture wascentrifuged at 10,000×g for 5 minutes, and the resulting supernatant wascombined with the first supernatant. Next, the supernatant was passedthrough Ultrafree-MC column (manufactured by Millipore) and a partthereof (10 μl) was subjected to HPLC. The Ultrafree-MC column was usedaccording to the method described in the manufacture's instructionsattached thereto.

[0538] The HPLC was carried out using TSK-gel ODS-80Ts Column (4.6×300mm; manufactured by TOSOH) as a column and 7% methanol-containing 0.02mol/l ammonium acetate buffer (pH 4.0) as an eluant at an elutiontemperature of 50° C. and a flow rate of 1 ml/min.

[0539] The product was detected by using a fluorescencespectrophotometer FP-920 (manufactured by JASCO Corporation) (excitationwavelength: 330 nm, radiation wavelength: 420 nm).

[0540] The product was identified by using its coincidence of elutiontime with that of a standard sugar chain as the marker.2-Aminobenzamide-labeled GlcNAcβ1-3Galβ1,4-GlcNAcβ1-3Galβ1-4Glc was usedas the standard sugar chain.

[0541] The product was determined by using a 2-aminobenzamide-labeledglucose polymer (manufactured by Oxford Glycoscience) as the standardand comparing the fluorescence intensities.

[0542] As a result of the activity measurement by using a cell extractof the stable transformant cells (Namalwa cells) transfected with acontrol plasmid (pAMo) or a G6 polypeptide expression plasmid (pAMo-G6),a ratio of the substrate (LNnT) converted into the product(GlcNAcβ1-3Galβ1,4-GlcNAcβ1-3Galβ1-4Glc) was almost 0% in the controlplasmid-transfected cells, while it was increased to 6.11% in the G6polypeptide expression plasmid-transfected cells. Also, a ratio of theother substrate (Galβ1-4GlcNAcβ1-3Galβ1,4-GlcNAc) converted into theproduct (GlcNAcβ1-3Galβ1,4-GlcNAcβ1-3Galβ1-4GlcNAc) was almost 0% in thecontrol plasmid-transfected cells, while it was increased to 3.95% inthe G6 polypeptide expression plasmid-introduced cells. That is, it wasfound that β1,3-N-acetylglucosaminyltransferase activity is increased inthe G6 polypeptide expression plasmid-introduced cells in comparisonwith the control plasmid-introduced cells.

[0543] Based on the above results, it was confirmed that the G6polypeptide is a novel β1,3-N-acetylglucosaminyltransferase. This resultshows that a sugar chain in which N-acetylglucosamine is linked viaβ1,3-linkage to the galactose residue present in the non-reducingterminal of a sugar chain can be synthesized by using the G6polypeptide.

[0544] (2) Activity Measurement Using Glycolipids as Substrates

[0545] β1,3-N-Acetylglucosaminyltransferase activity of G6 polypeptidewas measured by using glycolipids as substrates according to knownmethods [FEBS, 462, 289 (1999), J. Biol. Chem., 269, 14730-14737 (1994),J. Biol. Chem., 267, 23507 (1992), J. Biol. Chem., 267, 2994 (1992)].Specifically, the reaction was carried out at 37° C. for 16 hours in 20μl of reaction solution [150 mmol/l sodium cacodylate (pH 7.2), 10mmol/l UDP-GlcNAc (manufactured by SIGMA), 480 μmol/l UDP-[¹⁴C]GlcNAc(manufactured by Amersham), 0.4% Triton CF-54, 10 mmol/l MnCl₂, 250μmol/l glycolipid, the cell extract prepared in the above (1) (20 μg asprotein)]. As glycolipids, lactosylceramide (manufactured by SIGMA) andparagloboside (obtained from Yasunori Kushi at Tokyo Medical and DentalUniversity) were used. After completion of the reaction, 200 μl of 0.1mol/l KCl was added and lightly centrifuged to obtain the supernatant.The supernatant was passed through Sep-Pak plus C18 Cartridge (Waters),which had been washed once with 10 ml of methanol and equilibrated twicewith 10 ml of 0.1 mol/l KCl, to adsorb the glycolipid in the supernatantonto the cartridge. After washing the cartridge twice with 10 ml of purewater for HPLC, the adsorbed glycolipid was eluted with 5 ml ofmethanol. After concentrating the eluate to about 10 μl by using avacuum dryer, the concentrated solution was spotted on a TLC plate(HPTLC plate Silica gel 60: manufactured by MERCK) and developed using adeveloping solvent comprising a composition of chloroform:methanol:water(containing 0.2% CaCl₂)=65:35:8. After the development was carried outto a position 5 mm from the upper end of the TLC plate, the plate wasdried and then the radioactivity incorporated into the glycolipid wasmeasured using Bio Image Analyzer BAS 2000 (manufactured by Fuji PhotoFilm).

[0546] As a result of the activity measurement by using a cell extractof the stable transformant cells (Namalwa cells) transfected with acontrol plasmid (pAMo) or a G6 polypeptide expression plasmid (pAMo-G6),the activity was not detected in the cells transfected with the controlplasmid when lactosylceramide was used as the substrate, but theactivity was distinctively detected in the cells transfected with the G6polypeptide expression plasmid (conversion efficiency into the product0.125%). When paragloboside was used as a substrate, weak activity wasdetected even in the cells transfected with the control plasmid(conversion efficiency into the product 0.006%), but the activity wasdistinctively increased in the cells transfected with the G6 polypeptideexpression plasmid (conversion efficiency into the product 0.126%).Based on the above results and the results of the above (1), it wasconfirmed that the G6 polypeptide is a novelβ1,3-N-acetylglucosaminyltransferase which uses lactosylceramide andparagloboside as substrates. When the activity of G6 polypeptide usinglactosylceramide as a substrate was defined as 100%, the activity usingparagloboside as a substrate was 96%.

[0547] The above results show that a glycolipid in whichN-acetylglucosamine is added via β1,3-linkage to the galactose residuepresent in the non-reducing terminal of a sugar chain such as oflactosylceramide or paragloboside can be synthesized using the G6polypeptide.

EXAMPLE 8 Secreted Production of FLAG Peptide-fused G6 Polypeptide UsingInsect Cell as the Host

[0548] It was considered based on its primary sequence that the clonedβ1,3-N-acetylglucosaminyltransferase G6 comprises an N-terminalcytoplasmic region containing 14 amino acids, subsequentmembrane-binding region containing 18 amino acids rich in hydrophobicproperty, a stem region containing at least 12 amino acids and theremaining most part of the C-terminal region including a catalyticregion. Thus, secreted expression of the G6 polypeptide was carried outwherein the N-terminal cytoplasmic region containing 14 amino acids, themembrane-binding region containing 18 amino acids and a part of the stemregion (3 amino acids or 6 amino acids) was removed from the G6polypeptide, and an immunoglobulin signal sequence and FLAG peptide wasadded to the removed regions.

[0549] (1) Construction of a FLAG Peptide Fused-secretion Vector pAMoF2for Animal Cells

[0550] A secretion vector pAMoF2 was constructed for the secretedexpression of a polypeptide of interest fused with a FLAG peptide havingthe amino acid sequence represented by SEQ ID NO:13 at the N-terminal.

[0551] A HindIII-Asp718 fragment was obtained by digesting pAMo withHindIII and Asp718. As linkers for ligating HindIII digestion site andAsp718 digestion site, the following 6 DNAs [IgK-1 (nucleotide sequence:SEQ ID NO:14), IgK-2 (nucleotide sequence: SEQ ID NO:15), IgK-3(nucleotide sequence: SEQ ID NO:16), IgK-4 (nucleotide sequence: SEQ IDNO:17), IgK-5 (nucleotide sequence: SEQ ID NO:18), IgK-61 (nucleotidesequence: SEQ ID NO:19)] were synthesized. Also, the linker constructedby these DNAs encodes an immunoglobulin K signal sequence and FLAGpeptide, and respective restriction enzyme digestion sites of PmaCI,StuI and SnaBI are included therein. Each of these 6 DNAs wassynthesized using 380A DNA synthesizer manufactured by AppliedBiosystems. The synthesized DNAs were used after phosphorylation usingT4 polynucleotide kinase (manufactured by Takara Shuzo, the same shallapply hereinafter).

[0552] The plasmid pAMoF2 was constructed by ligating the 6phosphorylated synthetic DNAs obtained in the above with theHindIII-Asp718 fragment of about 8.7 kb.

[0553] (2) Construction of a Plasmid pAMoF2-i52S

[0554] As primers for PCR, a DNA having the nucleotide sequencerepresented by SEQ ID NO:20 (called C12-7) and a DNA having thenucleotide sequence represented by SEQ ID NO:21 (called C12-9) weresynthesized (it is possible to purchase from Sawady Technology). Theyare designed such that BamHI site and NotI site are included in C12-7and C12-9, respectively.

[0555] PCR was carried out using a kit manufactured by Takara Shuzo(GeneAmp™ DNA Amplification Reagent Kit with AmpliTaq™ Recombinant TaqPolymerase). The reaction solution was prepared according to the methodof the kit, and the reaction was carried out using a DNA thermal cycler(PERKIN ELMER CETUS DNA Thermal Cycler; Available from Takara Shuzo) bycarrying out 10 cycles, each cycle consisting of a reaction at 94° C.for 30 seconds, 65° C. for 1 minute and 72° C. for 2 minutes, and then afurther reaction was carried out at 72° C. for 7 minutes. As a template,10 ng of a plasmid pAMo-i (Japanese Published Unexamined PatentApplication No. 236398/99) was used. A DNA fragment of about 1.1 kb wasobtained by the PCR.

[0556] A plasmid pT7B-i52S No. 3 was constructed by ligating thePCR-amplified DNA fragment of about 1.1 kb to a T-vector pT7Blue(manufactured by Novagen).

[0557] Next, the plasmid pAMoF2-i52S was constructed.

[0558] A StuI-BanIII fragment of about 7.2 kb was obtained by digestingpAMoF2 with StuI and BanIII. A BanIII-NotI fragment of about 1.7 kb wasobtained by digesting pAMo with BanIII and NotI. A BamHI (bluntend)-NotI fragment of about 1.1 kb was obtained by digesting pT7B-i52SNo. 3 with BamHI, converting the 5′ protruding end formed by the BamHIdigestion to a blunt end using E. coli DNA polymerase I Klenow fragment,and then digesting it with NotI.

[0559] The plasmid pAMoF2-i52S was constructed by ligating the thusobtained StuI-BanIII fragment of about 7.2 kb, BanIII-NotI fragment ofabout 1.7 kb and BamHI (blunt end)-NotI fragment of about 1.1 kb.

[0560] (3) Construction of Plasmids pBS-G6sec1 and pBS-G6sec2

[0561] A DNA fragment encoding a region considered to have the catalyticactivity of G6 polypeptide (aspartic acid at position 36 to isoleucineat position 378 in SEQ ID NO:1 or isoleucine at position 39 toisoleucine at position 378 in SEQ ID NO:1) was subcloned.

[0562] PCR was carried out by using CB-543 having the nucleotidesequence represented by SEQ ID NO:22 and CB-545 having the nucleotidesequence represented by SEQ ID NO:23 as primers and the plasmid pBS-G6constructed in Example 3 as a template to prepare a DNA fragment ofabout 1.0 kb encoding a region of aspartic acid at posotion 36 toisoleucine at position 378 in SEQ ID NO:1. BamHI digestion site and XbaIdigestion site were included in CB-543 and CB-545, respectively. Theplasmid pBS-G6sec1 was constructed by digesting the PCR-amplifiedfragment with BamHI and XbaI and then inserted the digested productsbetween BamHI and XbaI of a vector pBluescript SK(−).

[0563] PCR was carried out by using CB-544 having the nucleotidesequence represented by SEQ ID NO:24 and CB-545 having the nucleotidesequence represented by SEQ ID NO:25 as primers and the plasmid pBS-G6constructed in Example 3 as a template to prepare a DNA fragment ofabout 1.0 kb encoding a region of isoleucine at position 39 toisoleucine at position 378 in SEQ ID NO:1. BamHI digestion site and XbaIdigestion site were ligated to CB-544 and CB-545, respectively. Theplasmid pBS-G6sec2 was constructed by digesting the PCR-amplifiedfragment with BamHI and XbaI and then inserting the digested productsbetween BamHI and XbaI of the vector pBluescript SK(−).

[0564] Platinum Pfx DNA polymerase (manufactured by GIBCO BRL) was usedin the PCR. As a template, 1 ng of the plasmid pBS-G6 was used. After 50μl the PCR solution was heated at 94° C. for 2 minutes, 25 cycles of thereaction was carried out, each cycle consisting of a reaction at 94° C.for 20 seconds, at 55° C. for 45 seconds and at 68° C. for 2 minutes.This process was carried out according to the manufacture's instructionsattached to the Platinum Pfx DNA polymerase kit.

[0565] The absence of errors by PCR was confirmed by sequencing DNAfragments inserted into pBS-G6sec1 and pBS-G6sec2.

[0566] (4) Production of a Recombinant Virus for Secreted Expression ofFLAG Peptide-fused G6 Polypeptide in Insect Cells

[0567] A recombinant virus was prepared by two steps, namely a step(step 1) in which a DNA encoding an objective protein is inserted into aspecial plasmid called transfer vector, and another step (step 2) inwhich a recombinant virus is obtained through homologous recombinationby co-transfection of the objective DNA-inserted transfer vectorprepared in the step 1 and a wild type virus in an insect cell. Thesteps were carried out by the following procedures using BaculoGoldStarter Kit (product No. PM-21001K) manufactured by Pharmingen accordingto the manufacture's instructions of the kit.

[0568] Step 1

[0569] Integration of a DNA Encoding FLAG Peptide-fused G6 Polypeptideinto a Transfer Vector

[0570] Plasmids pVL1393-F2G6sec1 and pVL1393-F2G6sec2 were constructedby inserting a DNA encoding FLAG peptide-fused G6 polypeptide betweenBamHI site and NotI site of a transfer vector pVL1393 (manufactured byPharmingen).

[0571] The pAMoF2-i52S prepared in the above (2) was digested withrestriction enzymes HindIII and NotI to obtain a HindIII-NotI fragmentof 1.2 kb.

[0572] A pVL1393 was digested with restriction enzymes BamHI and BstPIto obtain a BamHI-BstPI fragment of 3.2 kb.

[0573] A pVL1393 was digested with restriction enzymes NotI and BstPI toobtain a NotI-BstPI fragment of 6.4 kb.

[0574] As linkers for connecting BamHI site and HindIII site, the DNAfragments shown in SEQ ID NOs:26 and 27 were synthesized and the 5′-endswere phosphorylated by using T4 polynucleotide kinase.

[0575] pVL1393-F2i52S2 was constructed by ligating these 3 fragments andlinkers.

[0576] A BamHI-NotI fragment of about 9.6 kb was obtained by digestingpVL1393-F2i52S2 with BamHI and NotI. Also, a BamHI-NotI fragment ofabout 1.0 kb was obtained by digesting pBS-G6sec1 constructed in theabove (3) with restriction enzymes BamHI and NotI. A plasmidpVL1393-F2G6sec1 was constructed by ligating these 2 fragments.

[0577] A BamHI-NotI fragment of about 9.6 kb was obtained by digestingpVL1393-F2i52S2 with BamHI and NotI. Also, a BamHI-NotI fragment ofabout 1.0 kb was obtained by digesting pBS-G6sec2 constructed in theabove (3) with restriction enzymes BamHI and NotI. A plasmidpVL1393-F2G6sec2 was constructed by ligating these 2 fragments.

[0578] Step 2

[0579] Preparation of Recombinant Virus

[0580] A recombinant baculovirus was prepared by introducing a linearbaculovirus DNA (BaculoGold baculovirus DNA, manufactured by Pharmingen)and the plasmid prepared in the above (pVL1393-F2G6sec1 orpVL1393-F2G6sec2) into an insect cell Sf9 (manufactured by Pharmingen)cultured using TNM-FH insect medium (manufactured by Pharmingen)according to a lipofectin method [Protein, Nucleic Acid and Enzyme, 37,2701 (1992)]. A process for producing a pVL1393-F2G6sec1-derivedrecombinant baculovirus is described below. A pVL1393-F2G6sec2-derivedrecombinant baculovirus was also produced in the same manner.

[0581] In 12 μl of distilled water, 1 to 5 μg of pVL1393-F2G6sec1 and 15ng of the linear baculovirus DNA were dissolved, a mixture of 6 μl (6μg) of lipofectin (manufactured by GIBCO BRL) and 6 μl of distilledwater was added thereto, and the resulting mixture was allowed to standat room temperature for 15 minutes.

[0582] About 2×10⁶ of Sf9 cells were suspended in 2 ml of Sf900-IImedium (manufactured by GIBCO BRL) and were put into a cell cultureplastic dish of 35 mm in diameter, and a total volume of the mixedsolution of pVL1393-F2G6sec1, linear baculovirus DNA and lipofectin wasadded thereto, followed by culturing at 27° C. for 3 days.

[0583] From the culture, 1 ml of the culture supernatant containing therecombinant virus was recovered.

[0584] To the dish from which the culture supernatant was recovered, 1ml of the TNM-FH insect medium was newly added and further cultivationat 27° C. for 4 days was done. After the culturing, 1.5 ml of culturesupernatant containing the recombinant virus was further recovered inthe same manner.

[0585] (5) Preparation of a Recombinant Virus Solution

[0586] About 8×10⁶ of Sf9 cells were suspended in 5 ml of EX-CELL 400medium (manufactured by JRH), put into a 25 cm² flask (manufactured byGreiner) and allowed to stand at room temperature for 30 minutes toadhere the cells onto the flask, the supernatant was discarded, and then1 ml of EX-CELL 400 medium and 1 ml of a culture supernatant containingthe recombinant virus obtained in the above (4) were added to the flask.

[0587] After the addition, the cells and virus particles were thoroughlycontacted by gently shaking at room temperature for 1 hour, and thencultured at 27° C. for 4 days by adding 4 ml of the TNM-FH insectmedium.

[0588] The culture was centrifuged at 1,500×g for 10 minutes to obtainrecombinant virus-infected Sf9 cells and 5.5 ml of a recombinant virussolution.

[0589] About 2×10⁷ of Sf9 cells were suspended in 15 ml of EX-CELL 400medium, put into a 75 cm² flask (manufactured by Greiner) and allowed tostand at room temperature for 30 minutes to adhere the cells onto theflask, the supernatant was discarded, and then 5 ml of EX-CELL 400medium and 1 ml of the recombinant virus solution obtained in the abovewere added to the flask.

[0590] After the addition, the cells and virus particles were thoroughlycontacted by gently shaking at room temperature for 1 hour, and 10 ml ofthe TNM-FH insect medium was added thereto, followed by culturing at 27°C. for 4 days. The culture was centrifuged at 1,500×g for 10 minutes toobtain recombinant virus-infected Sf9 cells and 15 ml of a recombinantvirus solution.

[0591] A titer of the virus in the recombinant virus solution can becalculated by the following method (based on the manual of BaculoGoldStarter Kit manufactured by Pharmingen).

[0592] About 6×10⁶ of Sf9 cells are suspended in 4 ml of EX-CELL 400medium, put into a cell culture plastic dish of 60 mm in diameter andallowed to stand at room temperature for 30 minutes to adhere the cellsonto the dish, the supernatant is discarded, and then 400 μl of EX-CELL400 medium and 100 μl of the recombinant virus solution diluted to 10⁻⁴or 10⁻⁵ with EX-CELL 400 medium are added to the dish.

[0593] After the addition, the cells and virus particles are thoroughlycontacted by gently shaking the dish at room temperature for 1 hour.

[0594] After the contact, the medium is removed from the dish, and amixed solution of 2 ml of EX-CELL 400 medium (kept at 42° C.) containinga 2% low melting point agarose (Agarplaque Agarose; manufactured byPharmingen) and 2 ml of TNM-FH insect medium (kept at 42° C.) is pouredinto the dish and allowed to stand at room temperature for 15 minutes.

[0595] After the standing, the dish is wrapped with a vinyl tape toprevent drying, put into a sealable plastic container, and cultured at27° C. for 5 days.

[0596] After the culturing, 1 ml of PBS buffer containing 0.01% NeutralRed is added to the dish, followed by further culturing for 1 day, andthen the number of formed plaques is counted.

[0597] (6) Secreted Production and Purification of a FLAG Peptide-fusedG6 Polypeptide

[0598] Since the G6 polypeptide encoded by the plasmid pVL1393-F2G6sec1-or plasmid pVL1393-F2G6sec2-derived recombinant virus is expressed as asecreted fusion protein with FLAG peptide, it can be easily purifiedusing Anti-FLAG M1 Affinity Gel (manufactured by COSMO BIO).

[0599] The pVL1393-F2G6sec1-derived recombinant virus can secrete andproduce a polypeptide in which a presumed catalytic region of G6polypeptide (aspartic acid at position 36 to isoleucine at position 378in SEQ ID NO:1) is fused to the C-terminal of a FLAG peptide containing8 amino acids via a Gly residue and a Ser residue.

[0600] The pVL1393-F2G6sec2-derived recombinant virus can secrete andproduce a polypeptide in which a presumed catalytic region of G6polypeptide (isoleucine at position 39 to isoleucine at position 378 inSEQ ID NO:1) is fused to the C-terminal of a FLAG peptide containing 8amino acids via a Gly residue and a Ser residue.

[0601] About 2×10⁷ Sf21 cells were suspended in 15 ml of EX-CELL 400medium, put into a 75 cm² flask (manufactured by Greiner) and allowed tostand at room temperature for 30 minutes to adhesion the cells onto theflask, the supernatant was discarded, and 5 ml of EX-CELL 400 medium and1 ml of the recombinant virus solution obtained in the above (5) wereadded to the flask.

[0602] After the addition, the cells and virus particles were thoroughlycontacted by gently shaking at room temperature for 1 hour, and thencultured at 27° C. for 4 days by adding 10 ml of the TNM-FH insectmedium. The culture supernatant was obtained respectively at 15 ml bycentrifuging the culture at 1,500×g for 10 minutes.

[0603] Sodium azide, sodium chloride and calcium chloride were added to15 ml of the culture supernatant obtained in the above to give finalconcentrations of 0.1%, 150 mmol/l and 2 mmol/l, respectively, and 100μl of Anti-FLAG M1 Affinity Gel (manufactured by COSMO BIO) was addedthereto, followed by gently stirring at 4° C. overnight.

[0604] After the stirring, the Anti-FLAG M1 Affinity Gel was recoveredby centrifuging at 160×g for 10 minutes, and the gel was washed twicewith 1 ml of a buffer solution containing 50 mmol/l Tris-HCl (pH 7.4),150 mmol/l sodium chloride and 1 mmol/l calcium chloride.

[0605] After the washing, the gel was treated at 4° C. for 30 minutes byadding 30 μl of a buffer solution containing 50 mmol/l Tris-HCl (pH7.4), 150 mmol/l sodium chloride and 2 mmol/l EDTA to elute proteinsabsorbed onto the gel. Thereafter, a supernatant was obtained bycentrifuging at 160×g for 10 minutes.

[0606] To the gel, 30 μl of the buffer solution containing 50 mmol/lTris-HCl (pH 7.4), 150 mmol/l sodium chloride and 2 mmol/l EDTA wasadded again, and the mixture was treated at 4° C. for 10 minutes andcentrifuged at 160×g for 10 minutes to obtain a supernatant. Thereafter,by carrying out this process again, a total of three elution processeswere carried out and a total of 85 μl of the eluate was obtained.

[0607] To the eluate was added 1 mol/l calcium chloride to give a finalconcentration of 4 mmol/l.

[0608] After carrying out SDS-PAGE using 8 μl of the eluate prepared inthis manner, silver staining or Western blotting using an anti-FLAGantibody was carried. The silver staining was carried out using SilverStaining Kit Wako (manufactured by Wako Pure Chemical Industries). Themethod followed the manufacture's instructions of the kit. The Westernblotting using an anti-FLAG antibody was carried out by using YEASTAMINO-TERMINAL FLAG EXPRESSION KIT (manufactured by SIGMA). The methodfollowed the manufacture's instructions of the kit.

[0609] Results of the silver staining or Western blotting using ananti-FLAG antibody are shown in FIG. 3. FIG. 3A is a graph showing aresult of the silver staining carried out after purifying a secretedFLAG peptide-fused G6 polypeptide (G6sec1 or G6sec2) (lanes 3 and 5)from a culture supernatant (lanes 4 and 6) of Sf21 cell infected with arecombinant virus derived from a secreted FLAG peptide-fused G6polypeptide expression plasmid [pVL1393-F2G6sec1 (lanes 3 and 4) orpVL1393-F2G6sec2 (lanes 5 and 6)] using Anti-FLAG M1 Affinity Gel andsubsequently subjecting it to SDS polyacrylamide gel electrophoresis. Asa control, a sample (lane 1) was prepared in the same manner from aculture supernatant (lane 2) of Sf21 cell infected with a recombinantvirus derived from plasmid pVL1393. Each arrow indicates position andsize of the produced secreted G6 polypeptide.

[0610]FIG. 3B is a graph showing a result of Western blotting carriedout using an anti-FLAG peptide antibody after purifying a secreted FLAGpeptide-fused G6 polypeptide (G6sec1 or G6sec2) from a culturesupernatant of Sf21 cell infected with a recombinant virus derived froma secreted FLAG peptide-fused G6 expression plasmid [pVL1393-F2G6sec1(lane 2) or pVL1393-F2G6sec2 (lane 3)] using Anti-FLAG M1 Affinity Geland subsequently subjecting it to SDS polyacrylamide gelelectrophoresis. As a control, a sample (lane 1) was prepared in thesame manner from a culture supernatant of Sf21 cell infected with arecombinant virus derived from plasmid pVL1393. Each arrow indicatesposition and size of the produced secreted G6 polypeptide.

[0611] As a result, a broad band of approximately from 41 to 47 kD wasfound when an elution solution prepared from a culture supernatant ofSf21 infected with the pVL1393-F2G6sec1-derived recombinant virus wasused. The size of the main band was about 43 kD. The molecular weight ofthe polypeptide calculated from its amino acid sequence is 40.95 kD,because it is considered that the recombinant virus can secrete andproduce a polypeptide in which a presumed catalytic region of G6polypeptide (aspartic acid at position 36 to isoleucine at position 378in SEQ ID NO:1) is fused to the C-terminal of a FLAG peptide containing8 amino acids via a Gly residue and a Ser residue. The reason for thelarger molecular weight of the detected band than the calculated valueis considered to be due to addition of sugar chains. The polypeptide has4 possible N-linked sugar chain addition sites. The broad band indicatesthe presence of polypeptides having different numbers and sizes of addedsugar chains.

[0612] A broad band of approximately 41 to 47 kD was also found when anelution solution prepared from a culture supernatant of Sf21 infectedwith the pVL1393-F2G6sec2-derived recombinant virus was used. The sizeof the main band was about 43 kD. The molecular weight of thepolypeptide calculated from its amino acid sequence is 40.6 kD, becauseit is considered that the recombinant virus can secrete and produce apolypeptide in which a presumed catalytic region of G6 polypeptide(isoleucine at position 39 to isoleucine at position 378 in SEQ ID NO:1)is fused to the C-terminal of a FLAG peptide containing 8 amino acidsvia a Gly residue and a Ser residue. The reason for the larger molecularweight of the detected band than the calculated value is considered tobe due to addition of sugar chains. The polypeptide has 4 possibleN-linked sugar chain addition sites. The broad band indicates thepresence of polypeptides having different numbers and sizes of addedsugar chains.

[0613] On the other hand, the band was not detected when an elutionsolution prepared from a culture supernatant of Sf21 infected with therecombinant virus derived from the vector pVL1393.

[0614] Regarding the strength of bands detected by the Western blottingusing anti-FLAG antibody, when the strength of the band detected in theelution solution prepared from the recombinant virus derived frompVL1393-F2G6sec1 is defined as 1, the strength of the band detected inthe elution solution prepared from the recombinant virus derived frompVL1393-F2G6sec2 was 1.05.

[0615] Based on the above results, it was shown that FLAG peptide-fusedG6 polypeptide can be secreted and produced using insect cells and thatthe polypeptide can be easily purified using Anti-FLAG M1 Affinity Gel.

EXAMPLE 9 Activity Measurement Using FLAG Peptide-fused G6 PolypeptideProduced by Insect Cells

[0616] β1,3-N-Acetylglucosaminyltransferase activity of the FLAGpeptide-fused G6 polypeptide produced and purified in Example 8 wasmeasured by using various substrates.

[0617] (1) Activity Measurement Using 2-aminobenzene ModifiedOligosaccharides as the Substrate

[0618] β1,3-N-Acetylglucosaminyltransferase activity of the FLAGpeptide-fused G6 polypeptide secreted and produced in insect cells wasmeasured by using the eluate prepared in Example 8(6). The method ofExample 7 was used for the activity measurement.

[0619] As substrates, oligosaccharides [LNnT,Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc (hereinafter referred to as “2LN”),Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ13-3Galβ1-4GlcNAc (hereinafter referredto as “3LN”),Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc(hereinafter referred to as “4LN”),Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc(hereinafter referred to as “5LN”), orGal,1-4(SO₃-6)GlcNAcβ1-3Galβ1-4(SO₃-6)GlcNAc (hereinafter referred to as“L2L2”)] were labeled with 2-aminobenzamide and used.2-Aminobenzamide-labeling of oligosaccharides was carried out usingSIGMA 2AB glycan labeling kit (manufactured by Oxford Glycoscience)according to the manufacture's instructions of the kit. LNnT waspurchased from Oxford Glycosystems. Other oligosaccharides were obtainedfrom Seikagaku Corporation.

[0620] Specifically, the reaction was carried out at 37° C. for 16 hoursin 20 μl of an assay solution [150 mmol/l sodium cacodylate (pH 7.2), 50mmol/l UDP-GlcNAc (manufactured by SIGMA), 0.4% Triton CF-54, 10 mmol/lMnCl₂, 5 μmol/l 2-aminobenzamide-labeled sugar chain substrate, theabove purified enzyme solution], and then the product was detected byhigh performance liquid chromatography (HPLC, details will be describedbelow). As the enzyme, an eluate (1.5 μl) prepared from a culturesupernatant of Sf21 infected with the recombinant virus derived frompVL1393-F2G6sec1 or an eluate (1.4 μl) prepared from a culturesupernatant of Sf21 infected with the recombinant virus derived frompVL1393-F2G6sec2, prepared in Example 8(6), was used.

[0621] The assay solution after completion of the reaction was treatedat 100° C. for 5 minutes and then mixed with 50 μl of pure water forHPLC and centrifuged at 10,000×g for 5 minutes to obtain thesupernatant. To a tube containing the assay solution, 50 μl of purewater for HPLC was added again, the tube was washed, and suparnatant wasobtained by centrifugation at 10,000×g for 5 minutes and was combinedwith the first supernatant. Next, the supernatant was passed throughUltrafree-MC column (manufactured by Millipore) and a portion thereof(10 μl) was subjected to HPLC. The Ultrafree-MC column was usedaccording to the method described in the manufacture's instructionsattached thereto.

[0622] The HPLC was carried out using TSK-gel ODS-80Ts Column (4.6×300mm; manufactured by TOSOH) as a column and 0.02 mol/l ammonium acetatebuffer containing 7% methanol (pH 4.0) as an eluant at an elutiontemperature of 50° C. and a flow rate of 1 ml/min.

[0623] The product was detected using a fluorescence spectrophotometerFP-920 (manufactured by JASCO Corporation) (excitation wavelength: 330nm, radiation wavelength: 420 nm).

[0624] As a result that the activity measurement was carried out on asecretory enzyme produced and purified using thepVL1393-F2G6sec1-derived recombinant virus (called G6sec1) and anothersecretory enzyme produced and purified using thepVL1393-F2G6sec2-derived recombinant virus (called G6sec2), both showedthe β1,3-N-acetylglucosaminyltransferase activity. When G6sec1andG6sec2, respectively, were used, the conversion efficiencies of thesubstrate LNnT into the product were 9.85% and 11.2%, respectively. Aresult of the examination of substrate specificity using G6sec1 orG6sec2 is shown in Table 1 (Test 1 in Table 1). Relative activities whenthe activity measured using 2-aminobenzamide-labeled LNnT as thesubstrate is defined as 100% are shown in the table. On the other hand,the activity was not detected in a sample prepared using thepVL1393-derived recombinant virus (control virus).

[0625] Also, a purified enzyme (G6sec2) was again prepared using themethod shown in Example 8(6), its substrate specificity was againexamined, and the results are also shown in Table 1 (Test 2 in Table 1).The purified enzyme (280 μl) was prepared, from 30 ml of a culturesupernatant of Sf21 infected with the recombinant virus derived frompVL1393-F2G6sec2, and the assay was carried out using a 4 μl portionthereof. Relative activities when the activity measured using2-aminobenzamide-labeled LNnT as the substrate is defined as 100% areshown in the table. When LNnT was used as the substrate, the conversionefficiency into the product was 26.2%. TABLE 1 Substrate specificity ofβ1,3-N-acetylglucosaminyltransferase (G6) using 2-aminobenzamide-labeledoligosaccharides as substrates Relative activity (%) Test 1 Test 2Substrate name G6sec 1 G6sec 2 G6sec 2 LNnT 100 100 100 2LN 81.4 86.865.3 3LN 92.9 95.8 79.2 4LN 13.2 21.5 10.0 5LN 9.3 11.5 12.3 L2L2 5.9

[0626] As a result, it was found that poly-N-acetyllactosamine sugarchains (2LN, 3LN, 4LN and 5LN) also become substrates of the G6polypeptide. It was also found that the shorter poly-N-acetyllactosaminesugar chains (2LN and 3LN) are apt to become substrates of the G6polypeptide in comparison with the longer poly-N-acetyllactosamine sugarchains (4LN and 5LN). Furthermore, it was found that L2L2 which is asulfated poly-N-acetyllactosamine sugar chain also becomes a substrateof the G6 polypeptide.

[0627] The β1,3-N-acetylglucosaminyltransferase was also detected whenthe G6sec1- or G6sec2-adsorbed ANTI-FLAG M1 AFFINITY GEL (gel beforeelution of the enzyme) was used as an enzyme. An amount of the gelequivalent to the elution solution used in the aboveβ1,3-N-acetylglucosaminyltransferase activity measurement was used asthe enzyme. When G6sec1-adsorbed gel and G6sec1-adsorbed gel were used,the conversion efficiencies of LNnT used as the substrate were 6.64% and20.2%, respectively.

[0628] This result indicates that sugar chains can be synthesized evenwhen the enzyme is adsorbed to the gel. On the other hand, the activitywas not detected in the gel prepared in the same manner using thepVL1393-derived recombinant virus (control virus).

[0629] On the other hand, the G6sec1 and G6sec2 did not show the GlcNAcβ1,3-galactosyltransferase activity. The β1,3-galactosyltransferaseactivity measurement was carried out according to a conventional method[J. Biol. Chem., 274, 12499-12507 (1999)]. The reaction was carried outat 37° C. for 16 hours in 20 μl of an assay solution [14 mmol/l HEPES(pH 7.4), 75 μmol/l UDP-Gal (manufactured by SIGMA), 11 mmol/l MnCl₂, 88pmol/l sugar chain substrate, the above purified enzyme]. The amount ofthe enzyme used was the same as the amount used in the measurement ofβ1,3-N-acetylglucosaminyltransferase. As a substrate,2-aminobenzamide-labeled GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc(hereinafter referred to as “GlcNAc-2LN”) was used. The sugar chain wasprepared by treating 2-aminobenzamide-labeled LN3 with μ-galactosidaseand thereby removing the terminal galactose residue. Specifically, 100milli-units of μ-galactosidase (manufactured by Seikagaku Corporation)were added to about 60 nmol of 2-aminobenzamide-labeled LN3, thereaction was carried out at 37° C. for 16 hours, and the sugar chain wasprepared by inactivating β-galactosidase through its heat treatment at100° C. for 5 minutes.

[0630] It was shown by the above results that the FLAG peptide-fused G6polypeptide (G6sec1 or G6sec2) secreted and expressed in insect cellshas the β1,3-N-acetylglucosaminyltransferase activity but does not havethe β1,3-galactosyltransferase activity. From this result, it wasconfirmed again that the G6 polypeptide is not aβ1,3-galactosyltransferase but a β1,3-N-acetylglucosaminyltransferase.It was shown that the β1,3-N-acetylglucosaminyltransferase G6 can besecreted and produced in insect cells as a fusion protein with FLAGpeptide, and the produced fusion protein can be easily purified usingANTI-FLAG M1 AFFINITY GEL. It was shown that the produced fusion proteincan be used in synthesizing sugar chains such as apoly-N-acetyllactosamine sugar chain.

[0631] It was found that the productivity of the enzyme is high whenproduced by insect cells in comparison with the case of producing it byNamalwa cells.

[0632] (2) Activity Measurement Using Pyridylaminated Oligosaccharidesas Substrates

[0633] After carrying out the reaction at 37° C. for 14.5 hours in 30 μlof an assay solution [150 mmol/l sodium cacodylate (pH 7.2), 50 mmol/lUDP-GlcNAc (manufactured by SIGMA), 0.4% Triton CF-54, 10 mmol/l MnCl₂,50 μmol/l pyridylaminated sugar chain substrate, a purified enzymesolution (G6sec2)], the product was detected by HPLC. The purifiedenzyme (G6sec2) was obtained according to the method shown in Example8(6). The purified enzyme (280 μl) was prepared from 30 ml of a culturesupernatant of Sf21 infected with the recombinant virus derived frompVL1393-F2G6sec2, and the assay was carried out using 5 μl thereof. Assubstrates, LnNT, lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc(hereinafter referred to as “LNT”), lacto-N-fucopentaose II(Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc; hereinafter referred to as“LNFP-II”), lacto-N-fucopentaose III(Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc; hereinafter referred to as“LNFP-III”), lacto-N-fucopentaose V(Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc; hereinafter referred to as“LNFP-V”), and lacto-N-difucohexaose II(Galβ1-3(Fucα-4)GlcNAcβ1-3Galβ1-4(Fucαα1-3)Glc; hereinafter referred toas “LNDFH-II”) (all manufactured by Oxford Glycosystems) werefluorescently-labeled with aminopyridine and used. The substrates werefluorescently-labeled according to a conventional method [Agric. Biol.Chem., 54, 2169 (1990)].

[0634] After carrying out the reaction on each substrate using an assaysolution containing UDP-GlcNAc (saccharide donor) and an assay solutioncontaining no donor and subsequently analyzing by HPLC, peaks appearedonly in the assay solution containing UDP-GlcNAc was defined asproducts.

[0635] The assay solution after completion of the reaction was treatedat 100° C. for 5 minutes and then centrifuged at 10,000×g for 5 minutesto obtain a supernatant, and a part thereof (5 μl) was subjected toHPLC.

[0636] The HPLC was carried out using TSK-gel ODS-80Ts Column (4.6×300mm; manufactured by TOSOH) and 0.02 mol/l ammonium acetate buffer (pH4.0) as the eluant at an elution temperature of 50° C. and a flow rateof 0.5 ml/min.

[0637] Detection and determination of products were carried out using afluorescence spectrophotometer FP-920 (manufactured by JASCOCorporation) (excitation wavelength: 320 nm, radiation wavelength: 400nm).

[0638] Relative activities when the activity measured using LNnT as asubstrate is defined as 100% are shown in Table 2. TABLE 2 Substratespecificity of β1,3-N-acetylglucosaminyltransferase (G6) usingpyridylaminated oligosaccharides as substrates Substrate name Relativeactivity (%) LNnT 100 LNT 5.5 LNFP-II <0.05 LNFP-III 4.9 LNDFH-II <0.05LNFP-V 4.9

[0639] When LNnT was used as a substrate, the conversion efficiency ofthe substrate into the product was 12.8%. It was found that LNnT becomesa good substrate of G6 polypeptide (G6sec2), but LNT, LNFP-III andLNFP-V hardly become substrates. Furthermore, it was also found thatLNFP-II and LNDFH-II as oligosaccharides in which fucose is added viaα1,4-linkage to the GlcNAc residue present at position 2 from thenon-reducing terminal of LNT do not become substrates of the G6polypeptide.

[0640] (3) Activity Measurement Using Unlabeled Oligosaccharides asSubstrates

[0641] The glycosyltransferase reaction was carried out as follows. Thereaction was carried out at 37° C. for 16 hours in 40 μl of an assaysolution [50 mmol/l MOPS (pH 7.5), 5 mmol/l UDP-GlcNAc (manufactured bySIGMA), 5 mmol/l MnCl₂, 10 mmol/l sugar chain substrate, a purifiedenzyme solution (G6sec2)]. Next, the mixture was treated at 100° C. for5 minutes and then centrifuged at 10,000×g for 20 minutes to obtain asupernatant, and a part thereof was analyzed using HPAE/PAD (HighPerformance Anion Pulsed Amperometric Detection; manufactured byDIONEX). The method was specifically carried out according to aconventional method [Anal. Biochem., 189, 151-162 (1990), J. Biol.Chem., 273, 433-440 (1998)].

[0642] As a purified enzyme (G6sec2), 10 μl of the enzyme obtained inthe above (2) was used. As substrates, unlabeled oligosaccharidesμlactose (Galβ1-4Glc) and LNnT] were used.

[0643] After the reaction on each substrate using an assay solutioncontaining UDP-GlcNAc (saccharide donor) and an assay solutioncontaining no donor, analysis was carried out using HPAE/PAD and peaksappeared only in the assay solution containing UDP-GlcNAc were definedas products. The product was identified using coincidence of its elutiontime with that of standard sugar chain as the index. As standard sugarchains, GlcNAcβ1-3Galβ1,4Glc and GlcNAcβ1-3Galβ1,4GlcNAcβ1-3Galβ1-4Glcwere used.

[0644] When LNnT and lactose were used as substrates, the conversionefficiencies into the product were 0.48% and 0.30%, respectively. Whenthe activity using LNnT as a substrate is defined as 100%, the relativeactivity when lactose is used as a substrate is calculated to be 62.5%.Based on the above results, it was found that theβ1,3-N-acetylglucosaminyltransferase (G6) also uses lactose as a goodsubstrate in addition to LNnT.

[0645] (4) Activity Measurement Using Glycolipids

[0646] Using glycolipids as the substrate,β1,3-N-acetylglucosaminyltransferase activity of the G6 polypeptide wasmeasured according to known methods [FEBS, 462, 289 (1999), J. Biol.Chem., 269, 14730-14737 (1994), J. Biol. Chem., 267, 23507 (1992), J.Biol. Chem., 267, 2994 (1992)]. Specifically, the reaction was carriedout at 37° C. for 16 hours in 20 μl of a reaction solution [150 mmol/lsodium cacodylate (pH 7.2), 10 mmol/l UDP-GlcNAc (manufactured bySIGMA), 480 μmol/l UDP-[¹⁴C]GlcNAc (manufactured by Amersham), 0.4%Triton CF-54, 10 mmol/l MnCl₂, 250 μl/l glycolipid, purified enzyme(G6sec2)]. As the purified enzyme (G6sec2), 10 μl of the enzyme obtainedin the above (2) was used. As glycolipids, lactosylceramide,paragloboside, galactosylceramide (Type I) and galactosylceramide (TypeII) were used. Paragloboside was obtained from Yasunori Kushi at TokyoMedical and Dental University. Other glycolipids were purchased fromSIGMA. After completion of the reaction, 200 μl of 0.1 mol/l KCl wasadded and lightly centrifuged to obtain the supernatant. The supernatantwas passed through Sep-Pak plus C18 Cartridge (manufactured by Waters)which had been washed once with 10 ml of methanol and equilibrated bywashing twice with 10 ml of 0.1 mol/l KCl to adsorb the glycolipid inthe supernatant onto the cartridge. After washing the cartridge twicewith 10 ml of pure water for HPLC, the adsorbed glycolipid was elutedwith 5 ml of methanol. After concentrating the eluate to about 10 μlusing a vacuum dryer, the concentrated solution was plotted on a TLCplate (HPTLC plate Silica gel 60: manufactured by MERCK) and developedusing a developing solvent comprising a composition ofchloroform:methanol:water (containing 0.2% CaCl₂)=65:35:8. After thedevelopment was carried out to a position 5 mm from the upper end of theTLC plate, the plate was dried and then the radioactivity incorporatedinto the glycolipid was measured using Bio Image Analyzer BAS 2000(manufactured by Fuji Photo Film).

[0647] It was found that the FLAG-fused G6 polypeptide (G6sec2) useslactosylceramide and paragloboside as substrates. When lactosylceramideand paragloboside were used as the substrates, the conversionefficiencies were 5.39% and 25.2%, respectively. When the G6 polypeptideactivity using lactosylceramide as a substrate was defined as 100%, theactivity when paragloboside was used as a substrate was 468%.

[0648] On the other hand, the G6 polypeptide (G6sec2) did not showactivity for galactosylceramide (Type I) and galactosylceramide (TypeII).

[0649] Based on the above results, it was found that the G6 polypeptideis a β1,3-N-acetylglucosaminyltransferase which uses lactosylceramideand paragloboside as good substrates. In comparison with the result ofExample 7(2), it can be understood that paragloboside is more easilyused as a substrate than lactosylceramide in secreted G6 (G6sec2).Although it has been shown that β3GnT as a knownβ1,3-N-acetylglucosaminyltransferase uses paragloboside as a substratein vitro, its activity when lactosylceramide is used as a substrate islow [Glycobiology, 9, 1123 (1999)]. Accordingly, it is considered thatthe G6 polypeptide is a β1,3-N-acetylglucosaminyltransferase havingdifferent substrate specificity from that of β3GnT.

[0650] Furthermore, when the G6 polypeptide and GlcNAcβ1,4-galactosyltransferase are used in combination, it is possible tosynthesize, for example, paragloboside from lactosylceramide,neolactohexaosylceramide(Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide) fromparagloboside or neolactohexaosylceramide from lactosylceramide.

EXAMPLE 10 Synthesis of Glycolipids in Human Culture Cells Transfectedwith a G6 Polypeptide Expression Plasmid

[0651] Neutral glycolipids were extracted from Namalwa cells (108 cells)transfected with pAMo-G6 prepared in Example 6(1), Namalwa cells (10⁸cells) transfected with the vector pAMo and Namalwa cells transfectedwith no plasmid, and the compositions and expression levels ofglycolipids having N-acetyllactosamine structure in the non-reducingtermini of sugar chains were compared. The method was carried outaccording to known methods [Shujunsha, Cell Technology Supplement (SaiboKogaku Bessatsu), “Glycobiology Experiment Protocol (Glycobiology JikkenProtocol)”, Anal. Biochem., 223, 232 (1994)]. As antibodies, monoclonalantibodies capable of recognizing N-acetyllactosamine structure presentin the non-reducing termini of sugar chains [anti-N-acetyllactosamineantibodies 1B2 to 1B7: Arch. Biochem. Biophysics., 303, 125 (1993),Infect. Immun., 64, 4129 (1996), J. Comp. Neurol., 22, 607 (1988)] wereused.

[0652] Neutral glycolipids corresponding to 10⁷ cells were spotted on aTLC plate (HPTLC plate Silica gel60: manufactured by MERCK) anddeveloped using a developing solvent containing a composition ofchloroform:methanol:water (containing 0.2% CaCl₂)=60:35:8. As standardglycolipids, glucosylceramide (2 μg; hereinafter referred sometimes toas “GlcCer”), lactosylceramide (2 μg; hereinafter referred sometimes toas “LacCer”), lactotriaosylceramide (0.5 μg; hereinafter referredsometimes to as “Lc₃Cer”) and paragloboside (0.5 μg; hereinafterreferred sometimes to as “nLc₄Cer”) were spotted in the case of orcinolstaining, and paragloboside (nLc₄Cer) and neolactohexaosylceramide(hereinafter referred sometimes to as “nLc₆Cer”) in the case ofimmunostaining. Except for the standard glycolipids, two identicalplates were prepared and one of them was used in orcinol staining, andanother was used in immunostaining.

[0653] Each of the plates after development was soaked for 20 seconds ina solution containing a composition of isopropanol:water (containing0.2% CaCl₂):methanol=40:20:7. Next, the plate was covered with a PVDFmembrane (Immobilon: manufactured by Millipore) and a glass microfiberfilter (manufactured by ATTO), and glycolipids on the plate weretransferred to the PVDF membrane using TLC Thermal Blotter (manufacturedby ATTO) at 180° C. and at level 8 for 45 seconds. The PVDF membrane wassoaked for 1 hour in a solution which contains 5% skim milk andTBS-Tween 20 and then soaked for 2 hours in a solution which contains 5%skim milk and TBS-Tween 20 containing 1/100 volume ofanti-N-acetyllactosamine antibodies 1B2 to 1B7 (hybridoma culturesupernatants). The PVDF membrane was washed with TBS-Tween 20 threetimes and then soaked for 1 hour in a solution which contains 5% skimmilk and TBS-Tween 20 containing a horseradish peroxidase-labeledanti-mouse IgM antibody (0.4 μg/ml: manufactured by Jackson). The PVDFmembrane was washed three times with TBS-Tween 20, and then theantibody-bound glycolipids were detected using an ECL system(manufactured by Amersham Pharmacia).

[0654] Results of the orcinol staining are shown in FIG. 4A, and resultsof the immunostaining in FIG. 4B. Lane 1 in FIG. 4A shows development ofthe standard glycolipids (GlcCer, LacCer, Lc₃Cer and nLc₄Cer), and otherlanes show results of the orcinol staining performed on neutralglycolipids extracted from Namalwa cells transfected with no plasmid(lane 2), Namalwa cells transfected with the vector pAMo (lane 3) andNamalwa cells transfected with pAMo-G6 (lane 4) and developed on the TLCplate. Lane 1 in FIG. 4B shows development of the standard glycolipids(nLc₄Cer and nLc₆Cer), and other lanes show results of theimmunostaining by using an antibody capable of recognizingN-acetyllactosamine structure present in the non-reducing termini ofsugar chains (anti-N-acetyllactosamine antibodies 1B2) performed onneutral glycolipids extracted from Namalwa cells transfected with noplasmid (lane 2), Namalwa cells transfected with the vector pAMo (lane3) and Namalwa cells transfected with pAMo-G6 (lane 4) and developed onthe TLC plate. The arrows in B show positions of glycolipids (nLc₄Cerand nLc₆Cer) whose expression was increased in the Namalwa cellstransfected with pAMo-G6.

[0655] It was found that amounts of paragloboside (nLc₄Cer) andneolactohexaosylceramide(Galβ11-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide: abbreviated as“nLc₆Cer” in the drawing), which were glycolipids havingN-acetyllactosamine structure on the non-reducing termini of sugarchains, are increased in the Namalwa cells expressing G6 polypeptide, incomparison with the Namalwa cells transfected with the vector and theNamalwa cells transfected with no plasmid (FIG. 4B).

[0656] Based on the above results and the results of Example 9, it wasfound that the G6 polypeptide is involved in the synthesis ofglycolipids having N-acetyllactosamine structure in cells. As shown inExample 9, the G6 polypeptide can synthesize lactotriaosylceramide(GlcNAcβ1-3Galβ1-4Glc-ceramide) by using lactosylceramide(Galβ1-4Glc-ceramide) as the substrate. It is considered that, inappropriate cells expressing a β1,4-galactosyltransferase (e.g., Namalwacells), lactotriaosylceramide synthesized by the G6 polypeptide isconverted into paragloboside (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide) bythe further addition of galactose via β1,4-linkage by theβ1,4-galactosyltransferase. The above results indicate that a glycolipidin which N-acetyllactosamine is added via β1,3-linkage to the galactoseresidue present in the non-reducing terminal of a glycolipid sugar chainas a substrate can be synthesized by expressing the G6 polypeptide inappropriate cells which express glycolipid substrates of the G6polypeptide such as lactosylceramide and paragloboside. In addition, itis indicated that a glycolipid in which N-acetyllactosamine structure ora poly-N-acetyllactosamine sugar chain is added to the non-reducingterminal of a sugar chain on a glycolipid substrate can be synthesizedwhen a cell expresses an appropriate Galβ1,4-galactosyltransferase.

[0657] The above results show that the G6 polypeptide acts as alactosylceramide β1,3-N-acetylglucosaminyltransferase in cells and isinvolved in the synthesis of neolacto-series glycolipids andlacto-series glycolipids. Furthermore, it is considered that the G6polypeptide is also involved in the synthesis of a glycolipid having apoly-N-acetyllactosamine sugar chain, such as neolactohexaosylceramide,in cells by acting with a GlcNAc β1,4-galactosyltransferasesynergistically.

[0658] Although it has been shown that a known enzyme (β3GnT) shows weakβ1,3-N-acetylglucosaminyltransferase activity on lactosylceramide invitro, it has not been shown whether or not it can actually uselactosylceramide as a substrate in cells.

EXAMPLE 11 Examination of Expression Level of Transcripts of the G6 Genein Various Cells

[0659] Transcripts of the G6 gene were quantified by quantitative PCRaccording to conventional methods [Proc. Natl. Acad. Sci. USA, 87, 2725(1990), J. Biol. Chem., 269, 14730 (1994), Japanese Published UnexaminedPatent Application No. 181759/94].

[0660] Quantification of β-actin transcripts for correcting theexpression level of the gene was also performed in the same manner byquantitative PCR.

[0661] (1) Synthesis of Single-stranded cDNAs Derived from Various Cellsand Cell Lines

[0662] As cell lines, colon cancer cell lines (Colo201, Colo205, HCT-15,SW480, SW620, WiDR, LS180), lung cancer cell lines (AOI, EBC-1, PC-1,A549, ABC-1, EHHA-9, HAL8, HAL24, LX-1, PC-7, PC-9, PC-12, RERF-LC-MS),stomach cancer cell lines (KATOIII, MKN1, MKN7, MKN28, MKN45, MKN74,TMK1, HSC43), neuroblastoma cell lines (NAGAI, NB-9, SCCH-26, IMR-32,SK-N-SH), glioblastoma cell lines (A172, KG-1-C, YKG-1, T98G, U251,U-118-MG, G1-1), pancreatic cancer cell lines (Capan-1, Capan-2), aprostatic cancer cell line PC-3, a hepatic cancer cell line HepG2, anerythroleukemia cell line K562, granulocyte/monocyte cell lines (HL-60,U-937, U266), a T-cell line Jurkat and B-cell lines (Namalwa KJM-1,Namalwa, Daudi, Ramos, NALL-1, Raji) were used. Jurkat was obtained fromAichi Cancer Center. KATOIII and PC-9 were obtained from I B L Co., Ltd.Other cells can be obtained from Japanese Collection of ResearchBioresources (JCRB) cell bank (internet address,http://cellbank.nihs.go.jp/) or American Type Culture Collection.

[0663] Furthermore, polymorphonuclear leukocytes and mononuclearleukocytes were separated and isolated from peripheral blood of a healthadult using Polymorphprep™ as a kit manufactured by Nycomed Pharma. Thethus obtained mononuclear leukocytes were further separated intomonocytes and lymphocytes according to a conventional method [J.Immunol., 130, 706 (1977)].

[0664] A total RNA of each cell was prepared according to a conventionalmethod [Biochemistry, 18, 5294 (1977)]. Single-stranded cDNAs from totalRNAs were synthesized using a kit (SUPER™ Preamplification System:manufactured by BRL). Single-stranded cDNAs were synthesized from 5 μgof total RNAs in the case of cell lines, or from 1 μg of total RNAs inthe case of peripheral blood-derived leukocytes, and diluted with water50-folds and 10-folds, respectively, and used as templates of PCR.Oligo(dT) primers were used as primers.

[0665] (2) Synthesis of Single-stranded cDNAs Derived from Various HumanTissues

[0666] Single-stranded cDNAs were synthesized from mRNAs of varioushuman organs (manufactured by Clontech) in the same manner as in theabove (1). Single-stranded cDNAs were synthesized from 1 μg of mRNAs,diluted 240-folds and used as templates of PCR. Oligo(dT) primers wereused as primers. As mRNAs, mRNAs derived from the following 35 organswere used: 1. adrenal gland, 2. brain, 3. caudate nucleus, 4.hippocampus, 5. substantia nigra, 6. thalamus, 7. kidney, 8. pancreas,9. pituitary gland, 10. small intestine, 11. bone marrow, 12. amygdala,13. cerebellum, 14. corpus callosum, 15. fetal brain, 16. fetal kidney,17. fetal liver, 18. fetal lung, 19. heart, 20. liver, 21. lung, 22.lymph node, 23. mammary gland, 24. placenta, 25. prostate, 26. salivarygland, 27. skeletal muscle, 28. spinal cord, 29. spleen, 30. stomach,Galβ1. testis, 32. thymus, 33. thyroid, 34. trachea and 35. uterus.

[0667] Furthermore, a total RNA was prepared from the human colontissue, and a single-stranded cDNA was synthesized using 5 μg of thetotal RNA in the same manner as in the case of the above cell lines of(1).

[0668] (3) Preparation of Standard and Internal Control for QuantitativePCR

[0669] Standard and internal control were constructed using the pBS-G6constructed in Example 3(5) [cf. the following (a) and (b)].

[0670] β-Actin transcripts were determined in the same manner asdescribed in reports [J. Biol. Chem., 269, 14730 (1994), JapanesePublished Unexamined Patent Application No. 181759/94]. In determiningμ-actin transcripts, pUC119-ACT and pUC119-ACTd were converted intolinear DNAs by digesting with restriction enzymes (HindIII and Asp718)which cut out cDNA moieties and then used as a standard and internalcontrol, respectively [J. Biol. Chem., 269, 14730 (1994), JapanesePublished Unexamined Patent Application No. 181759/94]. After confirmingthat each plasmid was completely digested, they were used by seriallydiluting with water containing 1 μg/ml of yeast transfer RNAs.

[0671] (a) Preparation of a Standard for G6 Transcripts Determination

[0672] pBS-G6 constructed in Example 3(5) was converted into a linearDNA by digestion with a restriction enzyme (EcoRI) which cuts out the G6cDNA moiety and used as a standard for determination. After confirmingthat the plasmid was completely digested, it was used by seriallydiluting with water containing 1 μg/ml of yeast transfer RNAs.

[0673] (b) Preparation of Internal Control for G6 TranscriptsDetermination

[0674] pBS-G6 was digested with restriction enzymes MscI and BglII andthe linear DNAs were subjected to self-ligation to construct pBS-G6d inwhich 243 bp were deleted from the G6 cDNA. The pBS-G6d was convertedinto a linear DNA by digestion with a restriction enzyme (EcoRI) whichcuts out the G6 cDNA moiety and used as the internal control fordetermination. After confirming that the plasmid was completelydigested, it was used by serially diluting with water containing 1 μg/mlof yeast transfer RNAs.

[0675] (4) Determination of Transcripts of the G6 Gene UsingQuantitative PCR

[0676] Competitive-PCR as a quantitative PCR was carried out using thesingle-stranded cDNAs derived from cell lines and normal tissuesprepared in the above (1) and (2). As the primers for PCR, CB513 havingthe nucleotide sequence represented by SEQ ID NO:25 and CB515 having thenucleotide sequence represented by SEQ ID NO:28 were used for thedetection of G6 transcripts. For the detection of β-actin transcripts,CB53 having the nucleotide sequence represented by SEQ ID NO:29 and CB54having the nucleotide sequence represented by SEQ ID NO:30 were used.Also, a calibration curve was prepared by carrying out the PCR in thesame manner using the standard and internal control prepared in (3).

[0677] PCR was carried out by adding a DNA polymerase AmpliTaq Gold™(manufactured by Perkin Elmer) to 50 μl of a reaction solution [10mmol/l Tris-HCl (pH 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl₂, 0.2 mmol/ldNTP, 0.001% (w/v) gelatin, 0.2 μmol/l KCl gene specific primers]containing 10 1μl of the above single-stranded cDNAs and 10 μl (1 fg) ofthe internal control plasmid. The amount of the internal control plasmidor the amount of the standard plasmid was appropriately changeddepending on each tissue or cell.

[0678] PCR was carried out under the following conditions.

[0679] In quantification of G6 transcripts, heating was carried out at95° C. for 11 minutes, and 42 cycles were carried out, each cycleconsisting of a reaction at 95° C. for 30 seconds, at 65° C. for 1minute and at 72° C. for 2 minutes.

[0680] In quantification of the β-actin transcripts, heating was carriedout at 95° C. for 11 minutes, and 24 cycles were carried out, each cycleconsisting of a reaction at 95° C. for 1 minute, at 65° C. for 1 minuteand at 72° C. for 2 minutes.

[0681] A solution of 10 μl after the PCR was subjected toelectrophoresis using 1% agarose gel, and the gel was stained withethidium bromide and photographed. Staining intensity of the amplifiedfragment was measured by scanning the photograph using NIH Image System,and used as the amount of amplification. In order to carry out moreaccurate quantification of transcripts, similar PCR was carried out bychanging the number of cycles of the PCR. The amounts of the standardand internal control were changed depending on the number of cycles ofthe PCR.

[0682] Using 0.125 fg, 0.25 fg, 0.5 fg, 1 fg, 2 fg or 4 fg of thestandard prepared in the above (3) instead of the cell-derivedsingle-stranded cDNAs, PCR was carried out and the amounts of amplifiedfragments were measured, and a calibration curve was prepared byplotting the amount of cDNA against the amount of the amplifiedfragment.

[0683] When the above primers for quantification of G6 transcripts areused, a DNA fragment of 458 bp is amplified from G6 transcripts and theG6 standard, and a DNA fragment of 215 bp is amplified from the G6internal control (photograph in FIG. 5).

[0684] When the above primers for quantification of β-actin transcriptsare used, a DNA fragment of 649 bp is amplified from β-actin transcriptsand the β-actin standard, and a DNA fragment of 439 bp is amplified fromthe β-actin internal control (photograph in FIG. 5).

[0685] The amounts of G6 transcripts are shown in the histogram of FIG.5, Table 3-1 and Table 3-2, which are indicated as relative values whenthe amount of β-actin transcripts is defined as 1,000. TABLE 3-1Expression level of G6 transcripts in various cell lines G6 transcripts/β-actin transcripts Cell lines Cell type (× 1,000) Colo201 colonadenocarcinoma 0.43 Colo205 colon adenocarcinoma 3.08 HCT15 colonadenocarcinoma 0.43 SW480 colon adenocarcinoma 0.36 SW620 colonadenocarcinoma 0.50 WiDr colon adenocarcinoma 0.79 LS180 colonadenocarcinoma 0.27 AOI lung squamous cell carcinoma 0.24 EBC-1 lungsquamous cell carcinoma 1.04 PC-1 lung squamous cell carcinoma 0.57 A545lung adenocarcinoma 0.14 ABC-1 lung adenocarcinoma 0.18 EHHA-9 lungadenocarcinoma 0.17 HAL8 lung adenocarcinoma 2.51 HAL24 lungadenocarcinoma 0.41 LX-1 lung adenocarcinoma 2.07 PC-7 lungadenocarcinoma 7.24 PC-9 lung adenocarcinoma 0.44 PC-12 lungadenocarcinoma 0.57 RERF-LC-MS lung adenocarcinoma 3.62 KATOIII stomachcancer 3.37 MKN1 stomach cancer 0.35 MKN7 stomach cancer 1.29 MKN28stomach cancer 4.84 MKN45 stomach cancer 0.12 MKN74 stomach cancer 2.62TMK1 stomach cancer 0.16 HSC43 stomach cancer 1.06

[0686] TABLE 3-2 Expression level of G6 transcripts in various celllines G6 transcripts/ β-actin transcripts Cell lines Cell type (× 1,000)NAGAI neuroblastoma 0.07 NB-9 neuroblastoma 1.74 SCCH-26 neuroblastoma1.71 IRM32 neuroblastoma 1.63 SK-N-SH glioblastoma 1.20 A172glioblastoma 0.71 KG-1-C glioblastoma 0.04 YKG-1 glioblastoma 0.42 T98Gglioblastoma 0.30 U251 glioblastoma 1.38 U-118-MG glioblastoma 0.51 G1-1glioblastoma 0.12 Capan-1 pancreas adenocarcinoma 1.51 Capan-2 pancreasadenocarcinoma 0.19 PC-3 prostate adenocarcinoma 0.86 HepG2hepatocellular carcinoma 0.00 K562 clonic myelogenous leukemia 0.06U-937 histiocytic lymphoma 0.55 HL-60 promyelocytic leukemia 0.91Namalwa Burkitt's lymphoma 0.04 Namalwa KJM-1 Burkitt's lymphoma 0.38Daudi Burkitt's lymphoma 0.00 Raji Burkitt's lymphoma 0.02 RamosBurkitt's lymphoma 0.01 U266 mycloma 0.03 Jurkat acute T cell leukemia0.05 NALL-1 lymphoblastic leukemia 1.06

[0687] G6 transcripts were expressed in almost all of the 36 humantissues examined (FIG. 5). Expression level varied among tissues, and itwas expressed relatively frequently in the cerebellum, pituitary gland,trachea, lung, colon, placenta, testis and the like tissues.Furthermore, G6 transcripts were expressed also in monocytes andlymphocytes separated from human peripheral blood.

[0688] Regarding cell lines, it was expressed relatively frequently in acolon cancer cell line Colo205, lung cancer cell lines (EBC-1, HAL8,LX-1, PC-7, RERF-LC-MS), stomach cancer cell lines (KATOIII, MKN7,MKN28, MKN74, HSC43), neuroblastoma cell lines (NB-9, SCCH-26, IMR-32,SK-N-SH), a glioblastoma cell line (U251), a pancreatic cancer cell line(Capan-1) and a B-cell line (NALL-1) (Table 3-1, Table 3-2).

EXAMPLE 12 Structural Analysis of Chromosomal Gene Encoding G6Polypeptide

[0689] Currently, sequences of a large number of human chromosomal geneswhose functions are unknown are registered in data bases. Thus, bycomparing the sequence of G6 cDNA of the present invention withsequences of the human chromosomal genes registered in data bases, thereis a possibility to identify a human chromosomal gene encoding the G6polypeptide of the present invention (called G6 chromosomal gene) and toreveal its structure. When a chromosomal gene sequence which isidentical to the sequence of the G6 cDNA is registered, a promoterregion and exon and intron structure of the chromosomal gene encodingthe polypeptide of the present invention can be determined by comparingsequence of the cDNA and sequence of the chromosomal gene.

[0690] When the nucleotide sequence of human G6 cDNA (SEQ ID NO:2) wascompared with the sequences registered in GenBank [accessible from thehome page (http://www.ncbi.nlm.nih.gov/) of National Center forBiotechnology Information (NCBI) on internet], it was found that a partof the human chromosomal working draft sequence (131,716 bp) ofregistration No. AC025833 (published on May 30, 2000) (an assemblysequence comprising a sequence of positions 67,758 to 72,176: shown inSEQ ID NO:30) is identical to the nucleotide sequence of G6 cDNA. As aresult of the analysis, nucleotides of positions 1 to 3,721 in the G6cDNA sequence having the nucleotide sequence represented by SEQ ID NO:2is identical to nucleotides of positions 653 to 4,373 in SEQ ID NO:30.Thus, it was found that the G6 cDNA moiety shown in SEQ ID NO:2 isderived from one exon. It was considered that the sequence of AATAAApresent in positions 3,702 to 3707 in the G6 cDNA sequence representedby SEQ ID NO:2 is a polyadenylation signal. It was considered that anupstream sequence (654 bp) of the exon is a promoter region (includingtranscription controlling region) of the G6 chromosomal gene. When thepresence of consensus sequences in binding sequences of transcriptionfactors was analyzed on the promoter region (654 bp) by using MotifSearch Program of a sequence analyzing software GENETYX-MAC 10.1 whichwas prepared based on Transcription Factor Database [Nucleic AcidsResearch, 18, 1749 (1990), Trends in Biochemical Science, 16, 455(1991), Nucleic Acids Research, 20S, 2091 (1992), Nucleic AcidsResearch, 21S, 3117 (1993)], it was judged that the sequence has thepromoter region.

[0691] Since the sequence of registration No. AC025833 is derived fromthe human first chromosome, it was found that the G6 chromosomal gene ispositioned at the human first chromosome. The position of the G6chromosomal gene on the chromosome and its structure (promoter regionand exon region) were able to be specified for the first time by thepresent invention through the elucidation of the structure of the G6cDNA and function of the polypeptide encoded thereby. Furthermore, ithas not been found so far that the sequence of registration No. AC025833encodes a β1,3-N-acetylglucosaminyltransferase (G6 polypeptide).

INDUSTRIAL APPLICABILITY

[0692] The present invention provides a novel polypeptide havingGalβ1,3-N-acetylglucosaminyltransferase activity; a process forproducing the polypeptide; a DNA encoding the polypeptide; a recombinantvector containing the DNA; a transformant carrying the recombinantvector; an antibody which recognizes the polypeptide; a determinationmethod and an immunostaining method of the polypeptide of the presentinvention which use the antibody; a process using the polypeptide forproducing a sugar chain having GlcNAcβ1-3Gal structure, apoly-N-acetyllactosamine sugar chain and a complex carbohydratecontaining the above sugar chains; a process using a transformantcarrying the recombinant vector for producing a sugar chain having aGlcNAcβ1-3Gal structure, a poly-N-acetyllactosamine sugar chain and acomplex carbohydrate containing the above sugar chains; a method forscreening a substance which changes expression of a gene encoding thepolypeptide; a method for screening a substance which changesβ1,3-N-acetylglucosaminyltransferase activity of the polypeptide; amethod using the DNA or the antibody for diagnosing inflammatorydiseases and cancers (colon cancer, pancreatic cancer, gastric cancerand the like); and a method using the DNA for treating inflammatorydiseases and cancers (colon cancer, pancreatic cancer, gastric cancerand the like), a substance capable of changing expression of a geneencoding the polypeptide or a substance capable of changingβ1,3-N-acetylglucosaminyltransferase activity of the polypeptide.

[0693] Free text in Sequence Listing

[0694] SEQ ID NO:4—Synthetic DNA

[0695] SEQ ID NO:5—Synthetic DNA

[0696] SEQ ID NO:6—Synthetic DNA

[0697] SEQ ID NO:7—Synthetic DNA

[0698] SEQ ID NO:8—Synthetic DNA

[0699] SEQ ID NO:9—Synthetic DNA

[0700] SEQ ID NO:10—Synthetic DNA

[0701] SEQ ID NO:11—Synthetic DNA

[0702] SEQ ID NO:12—Synthetic DNA

[0703] SEQ ID NO:13—Amino acid sequence of FLAG peptide

[0704] SEQ ID NO:14—Synthetic DNA

[0705] SEQ ID NO:15—Synthetic DNA

[0706] SEQ ID NO:16—Synthetic DNA

[0707] SEQ ID NO:17—Synthetic DNA

[0708] SEQ ID NO:18—Synthetic DNA

[0709] SEQ ID NO:19—Synthetic DNA

[0710] SEQ ID NO:20—Synthetic DNA

[0711] SEQ ID NO:21—Synthetic DNA

[0712] SEQ ID NO:22—Synthetic DNA

[0713] SEQ ID NO:23—Synthetic DNA

[0714] SEQ ID NO:24—Synthetic DNA

[0715] SEQ ID NO:25—Synthetic DNA

[0716] SEQ ID NO:26—Synthetic DNA

[0717] SEQ ID NO:28—Synthetic DNA

[0718] SEQ ID NO:29—Synthetic DNA

[0719] SEQ ID NO:31—Synthetic DNA

1 32 1 378 PRT Homo sapiens 1 Met Arg Met Leu Val Ser Gly Arg Arg ValLys Lys Trp Gln Leu Ile 1 5 10 15 Ile Gln Leu Phe Ala Thr Cys Phe LeuAla Ser Leu Met Phe Phe Trp 20 25 30 Glu Pro Ile Asp Asn His Ile Val SerHis Met Lys Ser Tyr Ser Tyr 35 40 45 Arg Tyr Leu Ile Asn Ser Tyr Asp PheVal Asn Asp Thr Leu Ser Leu 50 55 60 Lys His Thr Ser Ala Gly Pro Arg TyrGln Tyr Leu Ile Asn His Lys 65 70 75 80 Glu Lys Cys Gln Ala Gln Asp ValLeu Leu Leu Leu Phe Val Lys Thr 85 90 95 Ala Pro Glu Asn Tyr Asp Arg ArgSer Gly Ile Arg Arg Thr Trp Gly 100 105 110 Asn Glu Asn Tyr Val Arg SerGln Leu Asn Ala Asn Ile Lys Thr Leu 115 120 125 Phe Ala Leu Gly Thr ProAsn Pro Leu Glu Gly Glu Glu Leu Gln Arg 130 135 140 Lys Leu Ala Trp GluAsp Gln Arg Tyr Asn Asp Ile Ile Gln Gln Asp 145 150 155 160 Phe Val AspSer Phe Tyr Asn Leu Thr Leu Lys Leu Leu Met Gln Phe 165 170 175 Ser TrpAla Asn Thr Tyr Cys Pro His Ala Lys Phe Leu Met Thr Ala 180 185 190 AspAsp Asp Ile Phe Ile His Met Pro Asn Leu Ile Glu Tyr Leu Gln 195 200 205Ser Leu Glu Gln Ile Gly Val Gln Asp Phe Trp Ile Gly Arg Val His 210 215220 Arg Gly Ala Pro Pro Ile Arg Asp Lys Ser Ser Lys Tyr Tyr Val Ser 225230 235 240 Tyr Glu Met Tyr Gln Trp Pro Ala Tyr Pro Asp Tyr Thr Ala GlyAla 245 250 255 Ala Tyr Val Ile Ser Gly Asp Val Ala Ala Lys Val Tyr GluAla Ser 260 265 270 Gln Thr Leu Asn Ser Ser Leu Tyr Ile Asp Asp Val PheMet Gly Leu 275 280 285 Cys Ala Asn Lys Ile Gly Ile Val Pro Gln Asp HisVal Phe Phe Ser 290 295 300 Gly Glu Gly Lys Thr Pro Tyr His Pro Cys IleTyr Glu Lys Met Met 305 310 315 320 Thr Ser His Gly His Leu Glu Asp LeuGln Asp Leu Trp Lys Asn Ala 325 330 335 Thr Asp Pro Lys Val Lys Thr IleSer Lys Gly Phe Phe Gly Gln Ile 340 345 350 Tyr Cys Arg Leu Met Lys IleIle Leu Leu Cys Lys Ile Ser Tyr Val 355 360 365 Asp Thr Tyr Pro Cys ArgAla Ala Phe Ile 370 375 2 3750 DNA Homo sapiens CDS (135)..(1271) 2aagaagactt ccatttttaa tgaccaacat gtattaagat ggacacctac tctacgaaac 60acgaagttct atggtctcga agaagcccgt gcctgtttaa aactgatcct aactaaaaac 120agacttgagt ggat atg aga atg ttg gtt agt ggc aga aga gtc aaa aaa 170 MetArg Met Leu Val Ser Gly Arg Arg Val Lys Lys 1 5 10 tgg cag tta att attcag tta ttt gct act tgt ttt tta gcg agc ctc 218 Trp Gln Leu Ile Ile GlnLeu Phe Ala Thr Cys Phe Leu Ala Ser Leu 15 20 25 atg ttt ttt tgg gaa ccaatc gat aat cac att gtg agc cat atg aag 266 Met Phe Phe Trp Glu Pro IleAsp Asn His Ile Val Ser His Met Lys 30 35 40 tca tat tct tac aga tac ctcata aat agc tat gac ttt gtg aat gat 314 Ser Tyr Ser Tyr Arg Tyr Leu IleAsn Ser Tyr Asp Phe Val Asn Asp 45 50 55 60 acc ctg tct ctt aag cac acctca gcg ggg cct cgc tac caa tac ttg 362 Thr Leu Ser Leu Lys His Thr SerAla Gly Pro Arg Tyr Gln Tyr Leu 65 70 75 att aac cac aag gaa aag tgt caagct caa gac gtc ctc ctt tta ctg 410 Ile Asn His Lys Glu Lys Cys Gln AlaGln Asp Val Leu Leu Leu Leu 80 85 90 ttt gta aaa act gct cct gaa aac tatgat cga cgt tcc gga att aga 458 Phe Val Lys Thr Ala Pro Glu Asn Tyr AspArg Arg Ser Gly Ile Arg 95 100 105 agg acg tgg ggc aat gaa aat tat gttcgg tct cag ctg aat gcc aac 506 Arg Thr Trp Gly Asn Glu Asn Tyr Val ArgSer Gln Leu Asn Ala Asn 110 115 120 atc aaa act ctg ttt gcc tta gga actcct aat cca ctg gag gga gaa 554 Ile Lys Thr Leu Phe Ala Leu Gly Thr ProAsn Pro Leu Glu Gly Glu 125 130 135 140 gaa cta caa aga aaa ctg gct tgggaa gat caa agg tac aat gat ata 602 Glu Leu Gln Arg Lys Leu Ala Trp GluAsp Gln Arg Tyr Asn Asp Ile 145 150 155 att cag caa gac ttt gtt gat tctttc tac aat ctt act ctg aaa tta 650 Ile Gln Gln Asp Phe Val Asp Ser PheTyr Asn Leu Thr Leu Lys Leu 160 165 170 ctt atg cag ttc agt tgg gca aatacc tat tgt cca cat gcc aaa ttt 698 Leu Met Gln Phe Ser Trp Ala Asn ThrTyr Cys Pro His Ala Lys Phe 175 180 185 ctt atg act gct gat gat gac atattt att cac atg cca aat ctg att 746 Leu Met Thr Ala Asp Asp Asp Ile PheIle His Met Pro Asn Leu Ile 190 195 200 gag tac ctt caa agt tta gaa caaatt ggt gtt caa gac ttt tgg att 794 Glu Tyr Leu Gln Ser Leu Glu Gln IleGly Val Gln Asp Phe Trp Ile 205 210 215 220 ggt cgt gtt cat cgt ggt gcccct ccc att aga gat aaa agc agc aaa 842 Gly Arg Val His Arg Gly Ala ProPro Ile Arg Asp Lys Ser Ser Lys 225 230 235 tac tac gtg tcc tat gaa atgtac cag tgg cca gct tac cct gac tac 890 Tyr Tyr Val Ser Tyr Glu Met TyrGln Trp Pro Ala Tyr Pro Asp Tyr 240 245 250 aca gcc gga gct gcc tat gtaatc tcc ggt gat gta gct gcc aaa gtc 938 Thr Ala Gly Ala Ala Tyr Val IleSer Gly Asp Val Ala Ala Lys Val 255 260 265 tat gag gca tca cag aca ctaaat tca agt ctt tac ata gac gat gtg 986 Tyr Glu Ala Ser Gln Thr Leu AsnSer Ser Leu Tyr Ile Asp Asp Val 270 275 280 ttc atg ggc ctc tgt gcc aataaa ata ggg ata gta ccg cag gac cat 1034 Phe Met Gly Leu Cys Ala Asn LysIle Gly Ile Val Pro Gln Asp His 285 290 295 300 gtg ttt ttt tct gga gagggt aaa act cct tat cat ccc tgc atc tat 1082 Val Phe Phe Ser Gly Glu GlyLys Thr Pro Tyr His Pro Cys Ile Tyr 305 310 315 gaa aaa atg atg aca tctcat gga cac tta gaa gat ctc cag gac ctt 1130 Glu Lys Met Met Thr Ser HisGly His Leu Glu Asp Leu Gln Asp Leu 320 325 330 tgg aag aat gct aca gatcct aaa gta aaa acc att tcc aaa ggt ttt 1178 Trp Lys Asn Ala Thr Asp ProLys Val Lys Thr Ile Ser Lys Gly Phe 335 340 345 ttt ggt caa ata tac tgcaga tta atg aag ata att ctc ctt tgt aaa 1226 Phe Gly Gln Ile Tyr Cys ArgLeu Met Lys Ile Ile Leu Leu Cys Lys 350 355 360 att agc tat gtg gac acatac cct tgt agg gct gcg ttt atc taa 1271 Ile Ser Tyr Val Asp Thr Tyr ProCys Arg Ala Ala Phe Ile 365 370 375 tagtacttga atgttgtatg ttttcactgtcactgagtca aacctggatg aaaaaaacct 1331 ttaaatgttc gtctataccc taagtaaaatgaggacgaaa gacaaatatt ttgaaagcct 1391 agtccatcag aatgtttctt tgattctagaagctgtttaa tatcacttat ctacttcatt 1451 gcctaagttc atttcaaaga atttgtatttagaaaaggtt tatattatta gtgaaaacaa 1511 aactaaaggg aagttcaagt tctcatgtaatgccacatat atacttgagg tgtagagatg 1571 ttattaagaa gttttgatgt tagaataattgcttttggaa aataccaaat gaacgtacag 1631 tacaacattt caaggaaatg aatatattgttagaccaggt aagcaagttt atttttgtta 1691 aagagcactt ggtggaggta gtaggggcagggaaaggtca gcataggaga gaaagttcat 1751 gaatctggta aaacagtctc ttgttcttaagaggagatgt agaaaaatgt gtacaatgtt 1811 attataaaca gacaaatcac gtcttaccacatccatgtag ctactggtgt tagagtcatt 1871 aaaatacctt tttttgcatc ttttttcaaagtttaatgtg aacttttaga aaagtgatta 1931 atgttgccct aatactttat atgtttttaatggatttttt tttaagtatt agaaaatgac 1991 acataacacg ggcagctggt tgctcatagggtccttctct agggagaaac cattgttaat 2051 tcaaataagc tgattttaat gacgttttcaactggttttt aaatattcaa tattggtctg 2111 tgtttaagtt tgttatttga atgtaatttacatagaggaa tataataatg gagagacttc 2171 aaatggaaag acagaacatt acaagcctaatgtctccata attttataaa atgaaatctt 2231 agtgtctaaa tccttgtact gattactaaaattaacccac tcctccccaa caaggtctta 2291 taaaccacag cactttgttc caagttcagagttttaaatt gagagcatta aacatcaaag 2351 ttataatatc taaaacaatt tatttttcatcaataactgt cagaggtgat ctttattttc 2411 taaatatttc aaacttgaaa acagagtaaaaaagtgatag aaaagttgcc agtttggggt 2471 taaagcattt ttaaagctgc atgttccttgtaatcaaaga gatgtgtctg agatctaata 2531 gagtaagtta catttatttt acaaagcaggataaaaatgt ggctataata cacactacct 2591 cccttcacta cagaaagaac taggtggtgtctactgctag ggagattata tgaaggccaa 2651 aataatgact tcagcaagag tgactgaactcactctaagg cctttgactg cagaggcacc 2711 tgttagggaa aatcagatgt ctcatataataaggtgatgt cggaaacacg caaaacaaaa 2771 cgaaaaaaga tttctcagta tacacaactgaatgatgata cttacaattt ttagcaggta 2831 gctttttaat gtttacagaa attttaatttttttctattt tgaaatttga ggcttgttta 2891 cattgcttag ataatttaga atttttaactaatgtcaaaa ctacagtgtc aaacattcta 2951 ggttgtagtt actttcagag tagatacagggttttagatc attacagttt aagttttctg 3011 accaattaaa aaaacataga gaacaaaagcatatttgacc aagcaacaag cttataatta 3071 atttttatta gttgattgat taatgatgtattgccttttg cccatatata ccctgtgtat 3131 ctatacttgg aagtgtttaa ggttgccattggttgaaaac ataagtgtct ctggccatca 3191 aagtgatctt gtttacagca gtgcttttgtgaaacaatta tttatttgct gaaagagctc 3251 ttctgaactg tgtcctttta atttttgcttagaatagaat ggaacaagtt taaatttcaa 3311 ggaaatatga aggcacttcc tttttttctaagaaggaagt tgctagatga ttccttcatc 3371 acacttactt aaagtactga gaagagtatctgtaaataaa agggttccaa ccttttaaaa 3431 aagaaggaaa aaactttttg gtgctccagtgtagggctat ctttttaaaa aatgtcaaca 3491 aagggaaaat taactatcag cttggatggtcacttgaata gaagatggtt atacacagtg 3551 ttattgttaa aattttttta ccttttggttggtttgcatc ttttttccat attgttaatt 3611 ttataccaaa atgttaaata tttgtattacttgaattttg ctcttgtatg gcaaaataat 3671 tagtgagttt aaaaaaaatc tatagtttccaataaacaac tgaaaaatta aaaaaaaaaa 3731 aaaaaaaaaa aaaaaaaaa 3750 3 738DNA Rattus norvegicus CDS (1)..(738) 3 c tac gag cga cgt tct gcc atc agaaag acg tgg ggc aac gag aat tac 49 Tyr Glu Arg Arg Ser Ala Ile Arg LysThr Trp Gly Asn Glu Asn Tyr 1 5 10 15 gtc cag tct cag ctc aac gcc aacatc aaa att ctg ttc gcg tta gga 97 Val Gln Ser Gln Leu Asn Ala Asn IleLys Ile Leu Phe Ala Leu Gly 20 25 30 act cct cat cca ctg aag gga aaa gagctg caa aaa aga ctg att tgg 145 Thr Pro His Pro Leu Lys Gly Lys Glu LeuGln Lys Arg Leu Ile Trp 35 40 45 gaa gat caa gtg tac cac gac ata att cagcaa gat ttc act gat tct 193 Glu Asp Gln Val Tyr His Asp Ile Ile Gln GlnAsp Phe Thr Asp Ser 50 55 60 ttc cac aat ctt act ttt aaa ttt ctt ctt cagttc ggc tgg gca aac 241 Phe His Asn Leu Thr Phe Lys Phe Leu Leu Gln PheGly Trp Ala Asn 65 70 75 80 acc ttt tgc cca cat gcc aga ttc ctg atg actgct gat gat gac ata 289 Thr Phe Cys Pro His Ala Arg Phe Leu Met Thr AlaAsp Asp Asp Ile 85 90 95 ttt atc cac atg cca aat ctc att gaa tac ctt caaggg ctg gag cag 337 Phe Ile His Met Pro Asn Leu Ile Glu Tyr Leu Gln GlyLeu Glu Gln 100 105 110 gtt gga gtt cga gac ttt tgg att ggt cac gtt caccga ggg ggc cct 385 Val Gly Val Arg Asp Phe Trp Ile Gly His Val His ArgGly Gly Pro 115 120 125 cct gtt aga gac aaa agt agc aag tac tat gtt ccctat gaa atg tac 433 Pro Val Arg Asp Lys Ser Ser Lys Tyr Tyr Val Pro TyrGlu Met Tyr 130 135 140 aag tgg cca gcc tac cct gac tat acc gcc ggt gctgcc tat gtc gtc 481 Lys Trp Pro Ala Tyr Pro Asp Tyr Thr Ala Gly Ala AlaTyr Val Val 145 150 155 160 tcc aac gat gta gct gcc aaa atc tat gag gcatca cag acg ctg aat 529 Ser Asn Asp Val Ala Ala Lys Ile Tyr Glu Ala SerGln Thr Leu Asn 165 170 175 tcc agc atg tac ata gac gat gtg ttc atg ggcctc tgc gcc aat aaa 577 Ser Ser Met Tyr Ile Asp Asp Val Phe Met Gly LeuCys Ala Asn Lys 180 185 190 gtg ggg gtc gtg cca cag gac cat gta ttt ttctct ggg gaa ggg aag 625 Val Gly Val Val Pro Gln Asp His Val Phe Phe SerGly Glu Gly Lys 195 200 205 att cct tac cat ccc tgc atc tat gaa aag atgata acg tct cat gga 673 Ile Pro Tyr His Pro Cys Ile Tyr Glu Lys Met IleThr Ser His Gly 210 215 220 cac tca caa gac cta cag gac ctc tgg gtg gaggcc aca gat cct aaa 721 His Ser Gln Asp Leu Gln Asp Leu Trp Val Glu AlaThr Asp Pro Lys 225 230 235 240 gtg aag gac att tcg aa 738 Val Lys AspIle Ser 4 24 DNA Artificial Sequence synthetic DNA 4 catcacagacactaaattca agtc 24 5 24 DNA Artificial Sequence synthetic DNA 5gcagtatatt tgaccaaaaa aacc 24 6 11 DNA Artificial Sequence synthetic DNA6 ctttagagca c 11 7 8 DNA Artificial Sequence synthetic DNA 7 ctctaaag 88 29 DNA Artificial Sequence synthetic DNA 8 ggggtaccat agatgcagggatgataagg 29 9 32 DNA Artificial Sequence synthetic DNA 9 ggggtaccgacttgaattta gtgtctgtga tg 32 10 30 DNA Artificial Sequence synthetic DNA10 ggggtaccat ctgtagcatt cttccaaagg 30 11 32 DNA Artificial Sequencesynthetic DNA 11 ggaattcccc tactctacga aacacgaagt tc 32 12 32 DNAArtificial Sequence synthetic DNA 12 ggaattccct ttcgtcctca ttttacttag gg32 13 8 PRT Artificial Sequence commercially available amino acidsequence 13 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 14 39 DNA ArtificialSequence synthetic DNA 14 agcttgccgc caccatgcat tttcaagtgc agattttca 3915 39 DNA Artificial Sequence synthetic DNA 15 gcttcctgct aatcagtgcctcagtcataa tgtcacgtg 39 16 36 DNA Artificial Sequence synthetic DNA 16gagattacaa ggacgacgat gacaaggcct acgtag 36 17 40 DNA Artificial Sequencesynthetic DNA 17 gaagctgaaa atctgcactt gaaaatgcat ggtggcggca 40 18 39DNA Artificial Sequence synthetic DNA 18 atctccacgt gacattatgactgaggcact gattagcag 39 19 35 DNA Artificial Sequence synthetic DNA 19gtacctacgt aggccttgtc atcgtcgtcc ttgta 35 20 30 DNA Artificial SequenceSynthetic DNA 20 cgcggatcct ccccacggtc cgtggaccag 30 21 36 DNAArtificial Sequence Synthetic DNA 21 atagtttagc ggccgcggaa gggctcagcagcgtcg 36 22 32 DNA Artificial Sequence Synthetic DNA 22 cgggatccgataatcacatt gtgagccata tg 32 23 32 DNA Artificial Sequence Synthetic DNA23 gctctagatg acagtgaaaa catacaacat tc 32 24 32 DNA Artificial SequenceSynthetic DNA 24 cgggatccat tgtgagccat atgaagtcat at 32 25 24 DNAArtificial Sequence Synthetic DNA 25 tcttatgact gctgatgatg acat 24 26 13DNA Artificial Sequence synthetic DNA 26 gatcatcgcg aga 13 27 13 DNAArtificial Sequence synthetic DNA 27 agcttctcgc gat 13 28 24 DNAArtificial Sequence synthetic DNA 28 ctttaggatc tgtagcattc ttcc 24 29 24DNA Artificial Sequence synthetic DNA 29 gatatcgccg cgctcgtcgt cgac 2430 4419 DNA Homo sapiens promoter (1)..(652) exon (653)..(4373) CDS(787)..(1923) 30 ttaaaaaaaa agaaaaaaga agtcttactc ttattcctgc cttgtctggggcaagcctta 60 atggattttt actgctgtga attttctttt cattgaagat tttgccttgatctatgtatc 120 tgctttcatc ctgaccatat tcaagtcagt atattcatga atgtacctgtttgtgaaatt 180 tgaacttaag tatacacgat tatagccgtt tgggaagctt ttttttttttttttttaaga 240 gtaggagtag aaaaaggtct ctgtactctg aatgggaaga cagtgtaaagcaattttttc 300 ccttttcctg tcctccttta aaaaaaataa acagccgtat gcctctgctaagtactaact 360 acctcatcac cttttgtgca gacagggcag gttacatttg gttttaaggaattaggaata 420 tgtttctttc cagcacctta gtaacccacg cgattgtgat tcttttctcttcttgactgt 480 gataggtggc atggaatatt cacatgggag agccgcatga ggccgcccaccacgcttcct 540 gaaggatgcc cgtgtggaag aattttgacg tgccagtgtc ctcgttctacagggtgttcc 600 attcttccgc aatctcagaa aaatgggact aaaagaaact attttgtaaaataagaagac 660 ttccattttt aatgaccaac atgtattaag atggacacct actctacgaaacacgaagtt 720 ctatggtctc gaagaagccc gtgcctgttt aaaactgatc ctaactaaaaacagacttga 780 gtggat atg aga atg ttg gtt agt ggc aga aga gtc aaa aaatgg cag 828 Met Arg Met Leu Val Ser Gly Arg Arg Val Lys Lys Trp Gln 1 510 tta att att cag tta ttt gct act tgt ttt tta gcg agc ctc atg ttt 876Leu Ile Ile Gln Leu Phe Ala Thr Cys Phe Leu Ala Ser Leu Met Phe 15 20 2530 ttt tgg gaa cca atc gat aat cac att gtg agc cat atg aag tca tat 924Phe Trp Glu Pro Ile Asp Asn His Ile Val Ser His Met Lys Ser Tyr 35 40 45tct tac aga tac ctc ata aat agc tat gac ttt gtg aat gat acc ctg 972 SerTyr Arg Tyr Leu Ile Asn Ser Tyr Asp Phe Val Asn Asp Thr Leu 50 55 60 tctctt aag cac acc tca gcg ggg cct cgc tac caa tac ttg att aac 1020 Ser LeuLys His Thr Ser Ala Gly Pro Arg Tyr Gln Tyr Leu Ile Asn 65 70 75 cac aaggaa aag tgt caa gct caa gac gtc ctc ctt tta ctg ttt gta 1068 His Lys GluLys Cys Gln Ala Gln Asp Val Leu Leu Leu Leu Phe Val 80 85 90 aaa act gctcct gaa aac tat gat cga cgt tcc gga att aga agg acg 1116 Lys Thr Ala ProGlu Asn Tyr Asp Arg Arg Ser Gly Ile Arg Arg Thr 95 100 105 110 tgg ggcaat gaa aat tat gtt cgg tct cag ctg aat gcc aac atc aaa 1164 Trp Gly AsnGlu Asn Tyr Val Arg Ser Gln Leu Asn Ala Asn Ile Lys 115 120 125 act ctgttt gcc tta gga act cct aat cca ctg gag gga gaa gaa cta 1212 Thr Leu PheAla Leu Gly Thr Pro Asn Pro Leu Glu Gly Glu Glu Leu 130 135 140 caa agaaaa ctg gct tgg gaa gat caa agg tac aat gat ata att cag 1260 Gln Arg LysLeu Ala Trp Glu Asp Gln Arg Tyr Asn Asp Ile Ile Gln 145 150 155 caa gacttt gtt gat tct ttc tac aat ctt act ctg aaa tta ctt atg 1308 Gln Asp PheVal Asp Ser Phe Tyr Asn Leu Thr Leu Lys Leu Leu Met 160 165 170 cag ttcagt tgg gca aat acc tat tgt cca cat gcc aaa ttt ctt atg 1356 Gln Phe SerTrp Ala Asn Thr Tyr Cys Pro His Ala Lys Phe Leu Met 175 180 185 190 actgct gat gat gac ata ttt att cac atg cca aat ctg att gag tac 1404 Thr AlaAsp Asp Asp Ile Phe Ile His Met Pro Asn Leu Ile Glu Tyr 195 200 205 cttcaa agt tta gaa caa att ggt gtt caa gac ttt tgg att ggt cgt 1452 Leu GlnSer Leu Glu Gln Ile Gly Val Gln Asp Phe Trp Ile Gly Arg 210 215 220 gttcat cgt ggt gcc cct ccc att aga gat aaa agc agc aaa tac tac 1500 Val HisArg Gly Ala Pro Pro Ile Arg Asp Lys Ser Ser Lys Tyr Tyr 225 230 235 gtgtcc tat gaa atg tac cag tgg cca gct tac cct gac tac aca gcc 1548 Val SerTyr Glu Met Tyr Gln Trp Pro Ala Tyr Pro Asp Tyr Thr Ala 240 245 250 ggagct gcc tat gta atc tcc ggt gat gta gct gcc aaa gtc tat gag 1596 Gly AlaAla Tyr Val Ile Ser Gly Asp Val Ala Ala Lys Val Tyr Glu 255 260 265 270gca tca cag aca cta aat tca agt ctt tac ata gac gat gtg ttc atg 1644 AlaSer Gln Thr Leu Asn Ser Ser Leu Tyr Ile Asp Asp Val Phe Met 275 280 285ggc ctc tgt gcc aat aaa ata ggg ata gta ccg cag gac cat gtg ttt 1692 GlyLeu Cys Ala Asn Lys Ile Gly Ile Val Pro Gln Asp His Val Phe 290 295 300ttt tct gga gag ggt aaa act cct tat cat ccc tgc atc tat gaa aaa 1740 PheSer Gly Glu Gly Lys Thr Pro Tyr His Pro Cys Ile Tyr Glu Lys 305 310 315atg atg aca tct cat gga cac tta gaa gat ctc cag gac ctt tgg aag 1788 MetMet Thr Ser His Gly His Leu Glu Asp Leu Gln Asp Leu Trp Lys 320 325 330aat gct aca gat cct aaa gta aaa acc att tcc aaa ggt ttt ttt ggt 1836 AsnAla Thr Asp Pro Lys Val Lys Thr Ile Ser Lys Gly Phe Phe Gly 335 340 345350 caa ata tac tgc aga tta atg aag ata att ctc ctt tgt aaa att agc 1884Gln Ile Tyr Cys Arg Leu Met Lys Ile Ile Leu Leu Cys Lys Ile Ser 355 360365 tat gtg gac aca tac cct tgt agg gct gcg ttt atc taa tagtacttga 1933Tyr Val Asp Thr Tyr Pro Cys Arg Ala Ala Phe Ile 370 375 atgttgtatgttttcactgt cactgagtca aacctggatg aaaaaaacct ttaaatgttc 1993 gtctataccctaagtaaaat gaggacgaaa gacaaatatt ttgaaagcct agtccatcag 2053 aatgtttctttgattctaga agctgtttaa tatcacttat ctacttcatt gcctaagttc 2113 atttcaaagaatttgtattt agaaaaggtt tatattatta gtgaaaacaa aactaaaggg 2173 aagttcaagttctcatgtaa tgccacatat atacttgagg tgtagagatg ttattaagaa 2233 gttttgatgttagaataatt gcttttggaa aataccaaat gaacgtacag tacaacattt 2293 caaggaaatgaatatattgt tagaccaggt aagcaagttt atttttgtta aagagcactt 2353 ggtggaggtagtaggggcag ggaaaggtca gcataggaga gaaagttcat gaatctggta 2413 aaacagtctcttgttcttaa gaggagatgt agaaaaatgt gtacaatgtt attataaaca 2473 gacaaatcacgtcttaccac atccatgtag ctactggtgt tagagtcatt aaaatacctt 2533 tttttgcatcttttttcaaa gtttaatgtg aacttttaga aaagtgatta atgttgccct 2593 aatactttatatgtttttaa tggatttttt tttaagtatt agaaaatgac acataacacg 2653 ggcagctggttgctcatagg gtccttctct agggagaaac cattgttaat tcaaataagc 2713 tgattttaatgacgttttca actggttttt aaatattcaa tattggtctg tgtttaagtt 2773 tgttatttgaatgtaattta catagaggaa tataataatg gagagacttc aaatggaaag 2833 acagaacattacaagcctaa tgtctccata attttataaa atgaaatctt agtgtctaaa 2893 tccttgtactgattactaaa attaacccac tcctccccaa caaggtctta taaaccacag 2953 cactttgttccaagttcaga gttttaaatt gagagcatta aacatcaaag ttataatatc 3013 taaaacaatttatttttcat caataactgt cagaggtgat ctttattttc taaatatttc 3073 aaacttgaaaacagagtaaa aaagtgatag aaaagttgcc agtttggggt taaagcattt 3133 ttaaagctgcatgttccttg taatcaaaga gatgtgtctg agatctaata gagtaagtta 3193 catttattttacaaagcagg ataaaaatgt ggctataata cacactacct cccttcacta 3253 cagaaagaactaggtggtgt ctactgctag ggagattata tgaaggccaa aataatgact 3313 tcagcaagagtgactgaact cactctaagg cctttgactg cagaggcacc tgttagggaa 3373 aatcagatgtctcatataat aaggtgatgt cggaaacacg caaaacaaaa cgaaaaaaga 3433 tttctcagtatacacaactg aatgatgata cttacaattt ttagcaggta gctttttaat 3493 gtttacagaaattttaattt ttttctattt tgaaatttga ggcttgttta cattgcttag 3553 ataatttagaatttttaact aatgtcaaaa ctacagtgtc aaacattcta ggttgtagtt 3613 actttcagagtagatacagg gttttagatc attacagttt aagttttctg accaattaaa 3673 aaaacatagagaacaaaagc atatttgacc aagcaacaag cttataatta atttttatta 3733 gttgattgattaatgatgta ttgccttttg cccatatata ccctgtgtat ctatacttgg 3793 aagtgtttaaggttgccatt ggttgaaaac ataagtgtct ctggccatca aagtgatctt 3853 gtttacagcagtgcttttgt gaaacaatta tttatttgct gaaagagctc ttctgaactg 3913 tgtccttttaatttttgctt agaatagaat ggaacaagtt taaatttcaa ggaaatatga 3973 aggcacttcctttttttcta agaaggaagt tgctagatga ttccttcatc acacttactt 4033 aaagtactgagaagagtatc tgtaaataaa agggttccaa ccttttaaaa aagaaggaaa 4093 aaactttttggtgctccagt gtagggctat ctttttaaaa aatgtcaaca aagggaaaat 4153 aaactatcagcttggatggt cacttgaata gaagatggtt atacacagtg ttattgttaa 4213 aatttttttaccttttggtt ggtttgcatc ttttttccat attgttaatt ttataccaaa 4273 atgttaaatatttgtattac ttgaattttg ctcttgtatg gcaaaataat tagtgagttt 4333 aaaaaaaatctatagtttcc aataaacaac tgaaaaatta tcatgagaag ggtatttaaa 4393 ctttttcatgaacattgctt atataa 4419 31 24 DNA Artificial Sequence synthetic DNA 31caggaaggaa ggctggaaga gtgc 24 32 245 PRT Rattus norvegicus 32 Tyr GluArg Arg Ser Ala Ile Arg Lys Thr Trp Gly Asn Glu Asn Tyr 1 5 10 15 ValGln Ser Gln Leu Asn Ala Asn Ile Lys Ile Leu Phe Ala Leu Gly 20 25 30 ThrPro His Pro Leu Lys Gly Lys Glu Leu Gln Lys Arg Leu Ile Trp 35 40 45 GluAsp Gln Val Tyr His Asp Ile Ile Gln Gln Asp Phe Thr Asp Ser 50 55 60 PheHis Asn Leu Thr Phe Lys Phe Leu Leu Gln Phe Gly Trp Ala Asn 65 70 75 80Thr Phe Cys Pro His Ala Arg Phe Leu Met Thr Ala Asp Asp Asp Ile 85 90 95Phe Ile His Met Pro Asn Leu Ile Glu Tyr Leu Gln Gly Leu Glu Gln 100 105110 Val Gly Val Arg Asp Phe Trp Ile Gly His Val His Arg Gly Gly Pro 115120 125 Pro Val Arg Asp Lys Ser Ser Lys Tyr Tyr Val Pro Tyr Glu Met Tyr130 135 140 Lys Trp Pro Ala Tyr Pro Asp Tyr Thr Ala Gly Ala Ala Tyr ValVal 145 150 155 160 Ser Asn Asp Val Ala Ala Lys Ile Tyr Glu Ala Ser GlnThr Leu Asn 165 170 175 Ser Ser Met Tyr Ile Asp Asp Val Phe Met Gly LeuCys Ala Asn Lys 180 185 190 Val Gly Val Val Pro Gln Asp His Val Phe PheSer Gly Glu Gly Lys 195 200 205 Ile Pro Tyr His Pro Cys Ile Tyr Glu LysMet Ile Thr Ser His Gly 210 215 220 His Ser Gln Asp Leu Gln Asp Leu TrpVal Glu Ala Thr Asp Pro Lys 225 230 235 240 Val Lys Asp Ile Ser 245

1. A polypeptide which comprises the amino acid sequence represented bySEQ ID NO:1.
 2. A polypeptide which comprises an amino acid sequence ofpositions 39 to 378 in the amino acid sequence represented by SEQ IDNO:1.
 3. A polypeptide which comprises an amino acid sequence in whichat least one amino acid in the amino acid sequence in the polypeptideaccording to claim 1 or 2 is deleted, substituted or added, and has aβ1,3-N-acetylglucosaminyltransferase activity.
 4. A polypeptide whichcomprises an amino acid sequence having 60% or more of homology with theamino acid sequence in the polypeptide according to claim 1 or 2, andhas a β1,3-N-acetylglucosaminyltransferase activity.
 5. The polypeptideaccording to claim 3 or 4, wherein theβ1,3-N-acetylglucosaminyltransferase activity is an activity to transferN-acetylglucosamine via β1,3-linkage to a galactose residue present inits non-reducing terminal of a sugar chain.
 6. The polypeptide accordingto any one of claims 3 to 5, wherein theβ1,3-N-acetylglucosaminyltransferase activity is an activity to transferN-acetylglucosamine via β1,3-linkage to a galactose residue present inits non-reducing terminal of a sugar chain of an acceptor selected fromi) galactose, N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc orlactose (Galβ1-4Glc), ii) an oligosaccharide having galactose,N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure in itsnon-reducing terminal, and iii) a complex carbohydrate having galactose,N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure in itsnon-reducing terminal.
 7. The polypeptide according to claim 6, whereinthe complex carbohydrate having galactose, N-acetyllactosamine,Galβ1-3GlcNAc or lactose structure in its non-reducing terminal islactosylceramide (Galβ1-4Glc-ceramide) or paragloboside(Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide).
 8. The polypeptide according toclaim 6 or 7, wherein the complex carbohydrate is a complex carbohydrateselected from a glycoprotein, a glycolipid, a proteoglycan, aglycopeptide, a lipopolysaccharide, a peptidoglycan and a glycoside inwhich a sugar chain is linked to a steroid compound.
 9. A sugar chainsynthesizing agent which comprises the polypeptide according to any oneof claims 1 to 8 as an active ingredient.
 10. A DNA which encodes thepolypeptide according to any one of claims 1 to
 8. 11. A DNA whichcomprises the nucleotide sequence represented by SEQ ID NO:2.
 12. A DNAwhich comprises a nucleotide sequence of positions 135 to 1268 in thenucleotide sequence represented by SEQ ID NO:2.
 13. A DNA whichcomprises a nucleotide sequence of positions 249 to 1268 in thenucleotide sequence represented by SEQ ID NO:2.
 14. A DNA whichhybridizes with a DNA comprising a nucleotide sequence complementary tothe nucleotide sequence in the DNA according to any one of claims 10 to13 under stringent conditions, and encodes a polypeptide havingβ1,3-N-acetylglucosaminyltransferase activity.
 15. The DNA according toclaim 14, wherein the Galβ1,3-N-acetylglucosaminyltransferase activityis an activity to transfer N-acetylglucosamine via β1,3-linkage to agalactose residue present in its non-reducing terminal of a sugar chain.16. The DNA according to claim 14 or 15, wherein theβ1,3-N-acetylglucosaminyltransferase activity is an activity to transferN-acetylglucosamine via β1,3-linkage to a galactose residue present inits non-reducing terminal of a sugar chain of an acceptor selected fromi) galactose, N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc orlactose (Galβ1-4Glc), ii) an oligosaccharide having galactose,N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure in itsnon-reducing terminal, and iii) a complex carbohydrate having galactose,N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure in itsnon-reducing terminal.
 17. The DNA according to claim 16, wherein thecomplex carbohydrate having galactose, N-acetyllactosamine,Galβ1-3GlcNAc or lactose structure in its non-reducing terminal islactosylceramide or paragloboside.
 18. The DNA according to claim 16 or17, wherein the complex carbohydrate is a complex carbohydrate selectedfrom a glycoprotein, a glycolipid, a proteoglycan, a glycopeptide, alipopolysaccharide, a peptidoglycan and a glycoside in which a sugarchain is linked to a steroid compound.
 19. A DNA which comprises anucleotide sequence complementary to the nucleotide sequence in the DNAaccording to any one of claims 10 to
 18. 20. A recombinant vector whichis obtainable by inserting the DNA according to any one of claims 10 to18 into a vector.
 21. A recombinant vector which is obtainable byinserting an RNA comprising a sequence homologous to the DNA accordingto any one of claims 10 to 18 into a vector.
 22. A transformant whichcomprises the recombinant vector according to claim 20 or
 21. 23. Thetransformant according to claim 22, wherein the transformant is atransformant selected from the group consisting of a microorganism, ananimal cell, a plant cell and an insect cell.
 24. The transformantaccording to claim 23, wherein the microorganism is a microorganismbelonging to the genus Escherichia.
 25. The transformant according toclaim 23, wherein the animal cell is an animal cell selected from thegroup consisting of a mouse myeloma cell, a rat myeloma cell, a mousehybridoma cell, a CHO cell, a BHK cell, an African green monkey kidneycell, a Namalwa cell, a Namalwa KJM-1 cell, a human fetal kidney celland a human leukemia cell.
 26. The transformant according to claim 23,wherein the plant cell is a plant cell selected from the groupconsisting of plant cells of tobacco, potato, tomato, carrot, soybean,rape, alfalfa, rice plant, wheat, barley, rye, corn or flax.
 27. Thetransformant according to claim 23, wherein the insect cell is an insectcell selected from the group consisting of Spodoptera frugiperda ovariancells, Trichoplusia ni ovarian cells and silkworm ovarian cells.
 28. Thetransformant according to claim 22, wherein the transformant is anon-human transgenic animal or transgenic plant.
 29. A process forproducing a polypeptide, which comprises culturing the transformantaccording to any one of claims 22 to 27 in a medium to produce andaccumulate the polypeptide according to any one of claims 1 to 8 in theculture, and recovering the polypeptide from the culture.
 30. A processfor producing a polypeptide, which comprises breeding the non-humantransgenic animal according to claim 28 to produce and accumulate thepolypeptide according to any one of claims 1 to 8 in the animal, andrecovering the polypeptide from the animal.
 31. The process according toclaim 30, wherein the accumulation is carried out in animal milk.
 32. Aprocess for producing a polypeptide, which comprises cultivating thetransgenic plant according to claim 28 to produce and accumulate thepolypeptide according to any one of claims 1 to 8 in the plant, andrecovering the polypeptide from the plant.
 33. A process for producing apolypeptide, which comprises synthesizing the polypeptide according toany one of claims 1 to 8 by an in vitro transcription-translation systemusing the DNA according to any one of claims 10 to
 18. 34. A process forproducing a sugar chain or complex carbohydrate by using the sugar chainsynthesizing agent according to claim 9 as an enzyme source, whichcomprises allowing a) the enzyme source, b) an acceptor selected from i)lactosylceramide (Galβ1-4Glc-ceramide) or paragloboside(Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide), ii) galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose(Galβ1-4Glc), iii) an oligosaccharide having galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose structurein its non-reducing terminal, and iv) a complex carbohydrate havinggalactose, N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure inits non-reducing terminal, and c) N-acetylglucosamine uridine5′-diphosphate (UDP-GlcNAc) to be present in an aqueous medium toproduce and accumulate a sugar chain or complex carbohydrate in whichN-acetylglucosamine is added via β1,3-linkage to a galactose residue ofthe acceptor in the aqueous medium, and recovering the sugar chain orcomplex carbohydrate from the aqueous medium.
 35. A process forproducing a galactose-added sugar chain or complex carbohydrate by usingthe N-acetylglucosamine-added sugar chain or complex carbohydrateobtained by the process according to claim 34 as an acceptor, whichcomprises allowing a) the acceptor, b) GlcNAcβ1,4-galactosyltransferase, and c) uridine 5′-diphosphate galactose(UDP-Gal) to be present in an aqueous medium to produce and accumulate areaction product in which galactose is added via β1,4-linkage to anN-acetylglucosamine residue at the non-reducing terminal of the acceptorin the aqueous medium, and recovering the galactose-added sugar chain orcomplex carbohydrate from the aqueous medium.
 36. A process forproducing a poly-N-acetyllactosamine sugar chain-added sugar chain orcomplex carbohydrate by using the sugar chain synthesizing agentaccording to claim 9 as an enzyme source, which comprises allowing a)the enzyme source, b) GlcNAc β1,4-galactosyltransferase, c) an acceptorselected from i) lactosylceramide (Galβ1-4Glc-ceramide) or paragloboside(Galβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide), ii) galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose(Galβ1-4Glc), iii) an oligosaccharide having galactose,N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose(Galβ1-4Glc) structure in its non-reducing terminal, iv) a complexcarbohydrate having galactose, N-acetyllactosamine, Galβ1-3GlcNAc orlactose structure in its non-reducing terminal and v) a sugar chain orcomplex carbohydrate obtained by the process according to claim 34 or35, d) uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc), and e)uridine 5′-diphosphate galactose (UDP-Gal) to be present in an aqueousmedium to produce and accumulate a reaction product in whichpoly-N-acetyllactosamine sugar chain is added to the non-reducingterminal of the acceptor in the aqueous medium, and recovering thepoly-N-acetyllactosamine sugar chain-added sugar chain or complexcarbohydrate from the aqueous medium.
 37. A process for producing asugar chain or complex carbohydrate, which comprises using thetransformant according to any one of claims 22 to 27 to produce andaccumulate a sugar chain comprising a saccharide selected from the groupconsisting of GlcNAcβ1-3Galβ1-4Glc-ceramide, a lacto-series glycolipid(a glycolipid having Galβ1-3GlcNAcβ1-3Galβ1-4Glc-ceramide as abackbone), a neolacto-series glycolipid (a glycolipid havingGalβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide as a backbone), a saccharide havingGlcNAcβ1-3Gal structure, a saccharide having GlcNAcβ1-3Galβ1-4GlcNAcstructure, a saccharide having GlcNAcβ1-3Galβ1-3GlcNAc structure, asaccharide having GlcNAcβ1-3Galβ1-4Glc structure, a saccharide having(Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc structure wherein n is 1 or moreand a saccharide having a (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structurewherein n is 1 or more, or a complex carbohydrate containing the sugarchain, and recovering the sugar chain or complex carbohydrate from theculture.
 38. A process for producing a sugar chain or complexcarbohydrate, which comprises using the non-human transgenic animal ortransgenic plant according to claim 28 to produce and accumulate a sugarchain comprising a saccharide selected from the group consisting ofGlcNAcβ1-3Galβ1-4Glc-ceramide, a lacto-series glycolipid (a glycolipidhaving Galβ1-3GlcNAcβ1-3Galβ1-4Glc-ceramide as a backbone), aneolacto-series glycolipid (a glycolipid havingGalβ1-4GlcNAcβ1-3Galβ1-4Glc-ceramide as a backbone), a saccharide havingGlcNAcβ1-3Gal structure, a saccharide having GlcNAcβ1-3Galβ1-4GlcNAcstructure, a saccharide having GlcNAcβ1-3Galβ1-3GlcNAc structure, asaccharide having GlcNAcβ1-3Galβ1-4Glc structure, a saccharide having(Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc structure wherein n is 1 or moreand a saccharide having (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structurewherein n is 1 or more, or a complex carbohydrate containing the sugarchain, and recovering the sugar chain or complex carbohydrate from theindividual.
 39. The process according to any one of claims 34 to 38,wherein the complex carbohydrate is selected from a glycoprotein, aglycolipid, a proteoglycan, a glycopeptide, a lipopolysaccharide, apeptidoglycan and a glycoside in which a sugar chain is linked to asteroid compound.
 40. The process according to claim 38, wherein theaccumulation is carried out in an animal milk.
 41. An oligonucleotidewhich has a sequence identical to continuos 5 to 120 nucleotides of thenucleotide sequence in the DNA according to any one of claims 10 to 19,or which is a derivative of the oligonucleotide.
 42. A method fordetermining expression level of a gene encoding the polypeptideaccording to any one of claims 1 to 8, which comprises using the DNAaccording to any one of claims 10 to 19, a partial fragment of the DNAor the oligonucleotide according to claim 41 by a hybridization method.43. A method for determining expression level of a gene encoding thepolypeptide according to any one of claims 1 to 8, which comprises usingthe oligonucleotide according to claim 41 by a polymerase chainreaction.
 44. A method for detecting an inflammation, cancer or tumormetastasis, which comprises using the method according to claim 42 or43.
 45. An agent for detecting an inflammation, cancer or tumormetastasis, which comprises the DNA according to any one of claims 10 to19, a partial fragment of the DNA or the oligonucleotide according toclaim
 41. 46. A method for diagnosing functional abnormality of a gene,which comprises detecting mutation of the gene which encodes thepolypeptide according to any one of claims 1 to
 8. 47. A method fordetecting mutation of a gene encoding the polypeptide according to anyone of claims 1 to 8, which comprises using the DNA according to any oneof claims 10 to 19, a partial fragment of the DNA or the oligonucleotideaccording to claim 41 by a hybridization method.
 48. A method fordetecting mutation of a gene encoding the polypeptide according to anyone of claims 1 to 8, which comprises using the oligonucleotideaccording to claim 41 by a polymerase chain reaction.
 49. A method forinhibiting transcription of a gene encoding the polypeptide according toany one of claims 1 to 8 or translation of mRNA thereof, which comprisesusing the oligonucleotide according to claim
 41. 50. An antibody whichrecognizes the polypeptide according to any one of claims 1 to
 8. 51. Amethod for immunologically detecting the polypeptide according to anyone of claims 1 to 8, which comprises using the antibody according toclaim
 50. 52. A method for immunohistostaining, which comprisesdetecting the polypeptide according to any one of claims 1 to 8 by usingthe antibody according to claim
 50. 53. An immunohistostaining agent,which comprises the antibody according to claim
 50. 54. A medicamentwhich comprises the polypeptide according to any one of claims 1 to 8.55. The medicament according to claim 54, which is a medicament fortreating, preventing and/or diagnosing an inflammatory disease, canceror tumor metastasis.
 56. A medicament which comprises the DNA accordingto any one of claims 10 to 19, a partial fragment of the DNA or theoligonucleotide according to claim
 41. 57. The medicament according toclaim 56, which is a medicament for treating, preventing and/ordiagnosing an inflammatory disease, cancer or tumor metastasis.
 58. Amedicament which comprises the recombinant vector according to claim 20or
 21. 59. The medicament according to claim 58, which is a medicamentfor treating, preventing and/or diagnosing an inflammatory disease,cancer or tumor metastasis.
 60. A medicament which comprises theantibody according to claim
 50. 61. The medicament according to claim60, which is a medicament for treating, preventing and/or diagnosing aninflammatory disease, cancer or tumor metastasis.
 62. A method forscreening a compound which changes aβ1,3-N-acetylglucosaminyltransferase activity possessed by thepolypeptide according to any one of claims 1 to 8, which measuringchanges in the β1,3-N-acetylglucosaminyltransferase activity of thepolypeptide caused by a sample to be tested by allowing the polypeptideto contact with the sample to be tested.
 63. The method according toclaim 62, wherein the β1,3-N-acetylglucosaminyltransferase activity isan activity to transfer N-acetylglucosamine via β1,3-linkage to agalactose residue present in the non-reducing terminal of a sugar chain.64. The method according to claim 62 or 63, wherein theβ1,3-N-acetylglucosaminyltransferase activity is an activity to transferN-acetylglucosamine via β1,3-linkage to a galactose residue present inits non-reducing terminal of a sugar chain of an acceptor selected fromi) galactose, N-acetyllactosamine (Galβ1-4GlcNAc), Galβ1-3GlcNAc orlactose (Galβ1-4Glc), ii) an oligosaccharide having galactose,N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure in thenon-reducing terminal, and iii) a complex carbohydrate having galactose,N-acetyllactosamine, Galβ1-3GlcNAc or lactose structure in thenon-reducing terminal.
 65. The method according to claim 62 or 63,wherein the complex carbohydrate having galactose, N-acetyllactosamine,Galβ1-3GlcNAc or lactose structure in the non-reducing terminal islactosylceramide or paragloboside.
 66. The method according to claim 64or 65, wherein the complex carbohydrate is a complex carbohydrateselected from a glycoprotein, a glycolipid, a proteoglycan, aglycopeptide, a lipopolysaccharide, a peptidoglycan and a glycoside inwhich a sugar chain is linked to a steroid compound.
 67. A method forscreening a compound which changes expression of a gene encoding thepolypeptide according to any one of claims 1 to 8, which comprisesallowing the polypeptide to contact with the sample to be tested, anddetermining paragloboside or poly-N-acetylglucosamine sugar chain byusing at least one selected from the group consisting of an antibodywhich recognizes paragloboside, an antibody or lectin which recognizespoly-N-acetylglucosamine sugar chain.
 68. A method for screening acompound which changes expression of a gene encoding the polypeptideaccording to any one of claims 1 to 8, which comprises allowing a cellexpressing the polypeptide to contact with a sample to be tested, anddetermining the polypeptide by using the antibody according to claim 50.69. A promoter DNA which controls transcription of a gene encoding thepolypeptide according to any one of claims 1 to
 8. 70. The promoter DNAaccording to claim 69, which is a promoter functioning in a cellselected from a leukocyte cell, a nerve cell, a tracheal cell, a lungcell, a colon cell, a placental cell, a neuroblastoma cell, aglioblastoma cell, a colon cancer cell, a lung cancer cell, a pancreaticcancer cell, a stomach cancer cell and a leukemia cell.
 71. The promoterDNA according to claim 69 or 70, which is a human-, rat- ormouse-derived promoter DNA.
 72. A method for screening a compound whichchanges efficiency of transcription by the promoter DNA according to anyone of claims 69 to 71, which comprises transforming an animal cell byusing a plasmid containing the promoter DNA and a reporter gene ligatedto the downstream of the promoter DNA, allowing the transformant tocontact with a sample to be tested, and determining the translatedproduct of the reporter gene.
 73. The method according to claim 72,wherein the reporter gene is a gene selected from a chloramphenicolacetyltransferase gene, a β-galactosidase gene, a β-lactamase gene, aluciferase gene and a green fluorescent protein gene.
 74. A compoundobtainable by the method according to any one of claims 62 to 68, 72 and73.
 75. A non-human knockout animal in which a deficiency or mutation isintroduced into a DNA encoding the polypeptide according to claims 1 to8.
 76. The knockout animal according to claim 75, wherein the non-humanknockout animal is a mouse.
 77. A method for controllingdifferentiation, mutual recognition and migration of a cell, whichcomprises introducing the DNA according to any one of claims 10 to 18,an RNA comprising a sequence homologous to the DNA or the recombinantvector according to claim 20 or 21 into a cell to express thepolypeptide according to any one of claims 1 to
 8. 78. The methodaccording to claim 77, wherein the cell is a cell selected from any oneof a blood cell, a nerve cell, a stem cell or a cancer cell.
 79. Amethod for accelerating differentiation of a promyelocyte into agranulocyte, which comprises introducing the DNA according to any one ofclaims 10 to 18, an RNA comprising a sequence homologous to the DNA orthe recombinant vector according to claim 20 or 21 into a promyelocyteto express the polypeptide according to any one of claims 1 to 8.